Plant transformation vector: Difference between revisions

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'''Plant transformation vectors''' are [[plasmid]]s that have been specifically designed to facilitate the generation of [[transgenic plants]]. The most commonly used plant transformation vectors are termed [[binary vectors|T-DNA binary vectors]] becauseand ofare theiroften ability to replicatereplicated in both ''[[E. coli]]'', a common lab [[Bacteria|bacterium]], and ''[[Agrobacterium tumefaciens]]'', a plant-virulent bacterium used to insert the [[recombinant (customized) DNA]] into plants.

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Plant Transformationtransformation vectors contain three key elements;:

'''Plant transformation vectors''' are [[plasmid]]s that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed [[binary vectors]] because of their ability to replicate in both ''[[E. coli]]'', a common lab bacterium, and ''[[Agrobacterium tumefaciens]]'', a bacterium used to insert the recombinant (customized) DNA into plants.

Plant Transformation vectors contain three key elements;

* Plasmids Selection (creating a custom circular strand of DNA)

* Plasmids Replication (so that it can be easily worked with)

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==Steps in plant transformation==

A custom DNA plasmid sequence can be created and replicated in various ways, but generally, all methods share the following processes:

Propagate binary vector in ''E. coli''

Plant transformation using plasmids begins 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.

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

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, which contains the foreign gene, becomes integrated into the plant cell genome. In each infected cell, the T-DNA is integrated at a different site in the genome.

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

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

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 ===

Foreign DNA inserted

* Foreign DNA inserted

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

* [[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 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 | last=Goldstein | first=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| 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>

* Transformation DNA fed to rodents ends up in their [[phagocyte]]s and rarely in other cells. Specifically, this isrefers to bacterial and [[M13 bacteriophage|M13]] DNA. (This preferential accumulation in phagocytes is thought to be real and not a detection artifact,artefact since these DNA extentssequences 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 | lastlast1=Goldstein | firstfirst1=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 | s2cid=40454719 }}</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 | bibcode=2008AnRPB..59..771L }}</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==

WhenA theselector bacteriagene withcan thebe desired,used implantedto genedistinguish aresuccessfully grown,genetically theymodified arecells madefrom containingunmodified a selectorones. AThe selector gene is aintegrated wayinto tothe isolateplasmid andalong distinguishwith the desired cells. Atarget gene, that makesproviding the cells resistantwith resistance to an [[antibiotic]], such as the antibiotics [[kanamycin]], [[ampicillin]], [[spectinomycin]] or [[tetracyclintetracycline]], is an easy selector to use. The desired cells, (along with any other organisms growing within the culture), can be treated with an antibiotic, allowing only the desiredmodified cells to survive while other organisms cannot. The antibiotic gene is not usually transferred to the plant cell but instead remains within the bacterial cell.

==Plasmids replication==

[[Plasmids]] replicate to produce many plasmid molecules in each host bacterial cell. The number of copies of each plasmid in a bacterial cell is determined by the [[replication origin]]. , Thiswhich is the position within the plasmidsplasmid molecule where DNA replication is initiated. Most [[binary vectors]] have a higher number of plasmidsplasmid copies when they replicate in ''[[E. coli]];'' however, the [[Plasmid copy number|plasmid copy-number]] is usually lesslower when the plasmid is resident within ''[[Agrobacterium tumefaciens]]''.

Plasmids can also be replicated inusing the [[polymerase chain reaction]] (PCR).

==T-DNA region==

T-DNA contains two types of genes: the [[Oncogene|oncogenic genes]], encoding for [[Enzyme|enzymes]] involved in the synthesis of [[Auxin|auxins]] and [[Cytokinin|cytokinins]] and responsible for [[tumor]] formation;, and the genes encoding for the synthesis of [[Opine|opines]]. These compounds, produced by the condensation between [[Amino acid|amino acids]] and sugars, are synthesized and excreted by the crown gall cells, and they are 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 cel 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).

The genes involved in opine [[catabolism]], T-DNA transfer from the bacterium to the plant cell and [[Bacterial conjugation|bacterium-bacterium plasmid conjugative transfer]] are located outside the T-DNA.<ref name=":0">{{Cite journal |last1=Hooykaas |first1=Paul J. J. |last2=Schilperoort |first2=Rob A. |date=1992-05-01 |title=Agrobacterium and plant genetic engineering |url=https://doi.org/10.1007/BF00015604 |journal=Plant Molecular Biology |language=en |volume=19 |issue=1 |pages=15–38 |doi=10.1007/BF00015604 |pmid=1600167 |bibcode=1992PMolB..19...15H |s2cid=36172990 |issn=1573-5028|url-access=subscription }}</ref><ref name=":1">{{Cite journal |last1=Zupan |first1=J. R. |last2=Zambryski |first2=P. |date=1995-04-01 |title=Transfer of T-DNA from Agrobacterium to the Plant Cell |url=https://doi.org/10.1104/pp.107.4.1041 |journal=Plant Physiology |volume=107 |issue=4 |pages=1041–1047 |doi=10.1104/pp.107.4.1041 |issn=0032-0889 |pmc=157234 |pmid=7770515}}</ref> 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 [[Protein|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 [[Operon|operons]] essential for the T-DNA transfer (virA, virB, virD, and virG) or for the increasing of transfer efficiency (virC and virE).<ref name=":0" /><ref name=":1" /><ref>{{Cite journal |last1=Jeon |first1=Geoung-A |last2=Eum |first2=Jin-seong |last3=Sim |first3=Woong Seop |date=1998-02-01 |title=The Role of Inverted Repeat (IR) Sequence of the virE Gene Expression in Agrobacterium tumefaciens pTiA6. |journal=Molecules and Cells |volume=8 |issue=1 |pages=49–53 |doi=10.1016/S1016-8478(23)13391-7 |pmid=9571631 |issn=1016-8478|doi-access=free }}</ref> Several 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 are involved in the synthesis and excretion of the b -1,2 [[glucan]],<ref>{{Cite journal |last1=Cangelosi |first1=G A |last2=Martinetti |first2=G |last3=Leigh |first3=J A |last4=Lee |first4=C C |last5=Theines |first5=C |last6=Nester |first6=E W |date=March 1989 |title=Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan |journal=Journal of Bacteriology |language=en |volume=171 |issue=3 |pages=1609–1615 |doi=10.1128/jb.171.3.1609-1615.1989 |issn=0021-9193 |pmc=209788 |pmid=2921245}}</ref> the {{not a typo|chvE}} required for the sugar enhancement of vir [[Gene induction|genes induction]] and [[Bacterial chemotaxis - general|bacterial chemotaxis]].<ref>{{Cite journal |last1=Ankenbauer |first1=R G |last2=Nester |first2=E W |date=November 1990 |title=Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides |journal=Journal of Bacteriology |language=en |volume=172 |issue=11 |pages=6442–6446 |doi=10.1128/jb.172.11.6442-6446.1990 |issn=0021-9193 |pmc=526831 |pmid=2121715}}</ref><ref>{{Cite journal |last1=Cangelosi |first1=G A |last2=Ankenbauer |first2=R G |last3=Nester |first3=E W |date=September 1990 |title=Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein. |journal=Proceedings of the National Academy of Sciences |language=en |volume=87 |issue=17 |pages=6708–6712 |doi=10.1073/pnas.87.17.6708 |doi-access=free |issn=0027-8424 |pmc=54606 |pmid=2118656|bibcode=1990PNAS...87.6708C }}</ref><ref name=":2">{{Citation |last1=Cangelosi |first1=Gerard A. |title=Bacterial Genetic Systems |date=1991 |pages=384–397 |url=https://doi.org/10.1016/0076-6879(91)04020-o |access-date=2024-03-09 |publisher=Elsevier |doi=10.1016/0076-6879(91)04020-o |last2=Abest |first2=Elaine |last3=Martinetti |first3=Gladys |last4=Nester |first4=Eugene W.|chapter=Genetic Analysis of Agrobacterium |series=Methods in Enzymology |volume=204 |pmid=1658565 |isbn=978-0-12-182105-0 |url-access=subscription }}</ref> The cell locus is responsible for the synthesis of [[cellulose]] fibrils.<ref>{{Cite journal |last=Matthysse |first=A G |date=May 1983 |title=Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection |journal=Journal of Bacteriology |language=en |volume=154 |issue=2 |pages=906–915 |doi=10.1128/jb.154.2.906-915.1983 |issn=0021-9193 |pmc=217544 |pmid=6302086}}</ref> The {{not a typo|pscA (exoC)}} locus is involved in the synthesis of both cyclic glucan and acid [[Glycan|succinoglycan]].<ref>{{Cite journal |last1=Cangelosi |first1=G A |last2=Hung |first2=L |last3=Puvanesarajah |first3=V |last4=Stacey |first4=G |last5=Ozga |first5=D A |last6=Leigh |first6=J A |last7=Nester |first7=E W |date=May 1987 |title=Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions |journal=Journal of Bacteriology |language=en |volume=169 |issue=5 |pages=2086–2091 |doi=10.1128/jb.169.5.2086-2091.1987 |issn=0021-9193 |pmc=212098 |pmid=3571162}}</ref><ref name=":2" /> The att locus is involved in the cell [[Surface protein|surface proteins]].<ref>{{Cite journal |last=Matthysse |first=Ann G. |date=October 1987 |title=Effect of Plasmid pSa and of Auxin on Attachment of Agrobacterium tumefaciens to Carrot Cells |journal=Applied and Environmental Microbiology |language=en |volume=53 |issue=10 |pages=2574–2582 |doi=10.1128/aem.53.10.2574-2582.1987 |issn=0099-2240 |pmc=204148 |pmid=16347473|bibcode=1987ApEnM..53.2574M }}</ref>

==References==