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Presentation on:
Plant transformation vectors and their
types
Presented to:
Dr. Hamid Manzoor
Presented by:
Hina Mushtaq …...
Institute of Molecular Biology & Biotechnology,
Bahauddin Zakariya University, Multan,
Pakistan.
Contents
 Plant transformation
 Vectors
 Types of vectors
 Plant transformation vectors
 Plasmids
 Viruses
 Bacteri...
Plant Transformation
”Transformation is the genetic alteration of
a cell resulting from the direct uptake and
incorporatio...
Steps of Plant Transformation
Plant Transformation Methods
1.Indirect method or vectored methods
oAgro bacterium-mediated transformation.
oVirus mediate...
Vector
“A DNA molecule used as a vehicle to carry
foreign genetic material into another cell.”
Types Of Vector:
-Plasmids....
Characteristics of vectors
 Origin of replication
 Self-replicating
 Bacterial selectable markers
 Gene constructs of ...
Vector classification
Cloning vectors
“Small piece of DNA into which a foreign DNA
fragment is inserted for cloning purpos...
In plants
 Plasmids
 Viruses
 Bacteriophages
 Cosmids
Plant Physiol. 2007 Dec; 145(4): 1118–1128.
doi: 10.1104/pp.107....
Plasmid
• Extra chromosomal DNA molecules.
•Self-replicating.
•Circular & Double stranded.
•Short sequence of DNA.
• Found...
Classification
Classification of plasmids
o Fertility plasmid
e.g. F plasmid of E. coli
o Col plasmid
e.g. ColE1 of E. coli
o Resistance ...
Based on the origin or source of plasmids
Two major classes :
i) Natural plasmids:
They occur naturally in prokaryotes
Exa...
Nomenclature of Plasmid
pBR322
p Plasmid
B Boliver
R Rodriguez
322 Number given to distinguish
http://blog.addgene.org/pla...
Advantages
Occur naturally in bacteria
Have different restriction sites.
Replicate completely independent of bacteria
...
Disadvantages
 Cannot accept large fragments
 Sizes range from 10-20 kb
 Standard methods of transformation are
ineffic...
Agrobacterium-mediated transformation
 Gram negative bacteria.
 Found in soil.
 Causes crown-gall disease.
 Ability to...
Agrobacterium tumefaciens
Recombinant Ti-plasmid
 Place target gene in T-DNA region.
 Recombinant T-DNA introduced into plants
http://www.csun.edu...
Plant genetic
engineering using T-
DNA vector
Method of screening
White-Blue screening
 Colonies with recombinant plasmid are white
 Colonies with non-recombinant plasmids are
blue.
For ...
Viral vectors
“Viruses which are used as gizmo by
molecular biologists to carry genetic
material into cells” are called vi...
Examples
1.Cauliflower mosaic virus based vectors.
2.Cowpea mosaic virus
3.Bean pod mottle virus (BPMV)
4.TMV based vector...
Characteristics of viral vectors
 Safety
 Low toxicity
 Stability
 Cell type specificity
http://www.ncbi.nlm.nih.gov/p...
Viruses are used in two ways
–Virus directly inserted into plant
–Virus indirectly inserted (bacteria)
http://www.ncbi.nlm...
Cauliflower mosaic virus
 DNA virus
 Infectious when simply rubbed on leaves
 Mechanical and aphid mediated transmissio...
Small insertions (10-30 bp) in various sites
abolished infectivity
The largest insert is 256-531 bp
CaMV genome can be ...
transcription
nucleus
35S RNA
19S RNA
translation
Reverse transcription
uncoating
Gene IV
Gene V
Gene III/IV
assembly
Incl...
Bacteriophage Lambda Vectors
 Viruses that can infect bacteria
 1000 times more efficient than plasmid vectors
 Clone D...
Steps
Advantages
 Fast processing ,low cost, high yield
 Good at targeting and entering cells
 Mostly target specific types o...
 Express proteins in plants for the
- Study of gene function
- Production of vaccines
- Study of metabolic engineering
- ...
Disadvantages
 Worst effects to plants by
–Producing severe disease
–Giving undesired products
–Affecting the plant adver...
Cosmid
 Derived from bacteriophage & plasmid
 Cohesive sites + plasmid = cosmid
 Less used for plant transformation
 C...
Cohesive ends or sticky ends
A single-stranded end of a linear
duplex DNA molecule which can
form hydrogen-bond with a
com...
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
Plant transformation vectors and their types
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Plant transformation vectors and their types

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Plant transformation vectors and their types

  1. 1. Presentation on: Plant transformation vectors and their types Presented to: Dr. Hamid Manzoor Presented by: Hina Mushtaq …………... BSBT-02 Zahra Naz ……………….. BSBT-04 07th May, 2015
  2. 2. Institute of Molecular Biology & Biotechnology, Bahauddin Zakariya University, Multan, Pakistan.
  3. 3. Contents  Plant transformation  Vectors  Types of vectors  Plant transformation vectors  Plasmids  Viruses  Bacteriophages – Advantages – Disadvantages
  4. 4. Plant Transformation ”Transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings.” or “Integration of gene into genome by means other than fusion of gametes” file:///G:/agri%20biotech/NPTEL%20__%20Biotechnology%20 -%20Plant%20Biotechnology.html
  5. 5. Steps of Plant Transformation
  6. 6. Plant Transformation Methods 1.Indirect method or vectored methods oAgro bacterium-mediated transformation. oVirus mediated. 2. Direct method. o Protoplast electroporation. o Protoplast polyethylene glycol method. o Gene gun method.
  7. 7. Vector “A DNA molecule used as a vehicle to carry foreign genetic material into another cell.” Types Of Vector: -Plasmids. -Viral vectors. -Cosmids. -Artificial chromosome. file:///G:/agri%20biotech/PLANT%20TRANSFORMATION%20VECTORS%20_%20What %20Is%20The%20Biotechnology.html
  8. 8. Characteristics of vectors  Origin of replication  Self-replicating  Bacterial selectable markers  Gene constructs of interest file:///G:/agri%20biotech/PLANT%20TRANSFORMATION%20VECTORS%20_%20What %20Is%20The%20Biotechnology.html
  9. 9. Vector classification Cloning vectors “Small piece of DNA into which a foreign DNA fragment is inserted for cloning purposes.” Expression vectors “Also known as an expression construct, is usually a plasmid or virus designed for protein expression in cells.” file:///G:/agri%20biotech/PLANT%20TRANSFORMATION%20VECTORS%20_%20What %20Is%20The%20Biotechnology.html
  10. 10. In plants  Plasmids  Viruses  Bacteriophages  Cosmids Plant Physiol. 2007 Dec; 145(4): 1118–1128. doi: 10.1104/pp.107.106104 PMCID: PMC2151730 Focus Issue on Vector Systems for Plant Research and Biotechnology Delivery of Multiple Transgenes to Plant Cells Mery Dafny-Yelin and Tzvi Tzfira
  11. 11. Plasmid • Extra chromosomal DNA molecules. •Self-replicating. •Circular & Double stranded. •Short sequence of DNA. • Found in prokaryotes. file:///G:/agri%20biotech/NPTEL%20__%20Biotechnology%20- %20Plant%20Biotechnology.html
  12. 12. Classification
  13. 13. Classification of plasmids o Fertility plasmid e.g. F plasmid of E. coli o Col plasmid e.g. ColE1 of E. coli o Resistance plasmid e.g. RP4 in Pseudomonas o Degradative plasmid e.g. TOL of P. putida o Virulence plasmid e.g. Ti plasmids of A. tumefaciens file:///C:/Users/%20/Documents/d.html
  14. 14. Based on the origin or source of plasmids Two major classes : i) Natural plasmids: They occur naturally in prokaryotes Example: ColE1. ii) Artificial plasmids: They are constructed in-vitro by re-combining selected segments of two or more plasmids. Example: pBR322. file:///C:/Users/%20/Documents/d.html
  15. 15. Nomenclature of Plasmid pBR322 p Plasmid B Boliver R Rodriguez 322 Number given to distinguish http://blog.addgene.org/plasmids-101-how-to-name-your-plasmid-in-3- easy-steps
  16. 16. Advantages Occur naturally in bacteria Have different restriction sites. Replicate completely independent of bacteria Genes are easily inserted into plasmids Easily transformed into bacteria https://answers.yahoo.com/question/index?qid=20061217124130AAsvKqk
  17. 17. Disadvantages  Cannot accept large fragments  Sizes range from 10-20 kb  Standard methods of transformation are inefficient https://answers.yahoo.com/question/index?qid=20061217124130AAsvKqk
  18. 18. Agrobacterium-mediated transformation  Gram negative bacteria.  Found in soil.  Causes crown-gall disease.  Ability to introduce DNA into plant. Contains - Ti-plasmid. - Ri-plasmid Curr Top Microbiol Immunol. 2014;375:155- 92. doi: 10.1007/82_2013_352. Plant viral vectors for delivery by Agrobacterium. Gleba YY, Tusé D, Giritch A
  19. 19. Agrobacterium tumefaciens
  20. 20. Recombinant Ti-plasmid  Place target gene in T-DNA region.  Recombinant T-DNA introduced into plants http://www.csun.edu/~hcbio027/biotechnology/lec2/PL/pl.htm
  21. 21. Plant genetic engineering using T- DNA vector
  22. 22. Method of screening
  23. 23. White-Blue screening  Colonies with recombinant plasmid are white  Colonies with non-recombinant plasmids are blue. For example: pUC19 Resistance to ampicilline. Contains portion of the lacZ which codes for beta-galactosidase.
  24. 24. Viral vectors “Viruses which are used as gizmo by molecular biologists to carry genetic material into cells” are called viral vectors.  Viral vectors are non-integrative as compared to bacterial vectors http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613721/
  25. 25. Examples 1.Cauliflower mosaic virus based vectors. 2.Cowpea mosaic virus 3.Bean pod mottle virus (BPMV) 4.TMV based vectors. 5.Potato virus X (PVX) 6.Bean yellow dwarf virus 7.Bacteriophage Lambda Vectors http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613721/
  26. 26. Characteristics of viral vectors  Safety  Low toxicity  Stability  Cell type specificity http://www.ncbi.nlm.nih.gov/pubmed/12828943
  27. 27. Viruses are used in two ways –Virus directly inserted into plant –Virus indirectly inserted (bacteria) http://www.ncbi.nlm.nih.gov/pubmed/12828943
  28. 28. Cauliflower mosaic virus  DNA virus  Infectious when simply rubbed on leaves  Mechanical and aphid mediated transmission  Up to 106 copies per cell within 3-4 weeks of infection in plant. Use of viral vectors for vaccine production in plants, M Carmen Cañizares, Liz Nicholson and George P Lomonossoff. John Innes Centre, Norwich, UK
  29. 29. Small insertions (10-30 bp) in various sites abolished infectivity The largest insert is 256-531 bp CaMV genome can be inserted into Ti vector
  30. 30. transcription nucleus 35S RNA 19S RNA translation Reverse transcription uncoating Gene IV Gene V Gene III/IV assembly Inclusion body (gene VI) Gene I CaMV activity in plant cell
  31. 31. Bacteriophage Lambda Vectors  Viruses that can infect bacteria  1000 times more efficient than plasmid vectors  Clone DNA fragments in range of 10,000 - 20,000 bps Bacteriophage. 2012 Oct 1; 2(4): 215–224. Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophages Two possible strategies for improving bacteriophage persistence for plant disease control Fanny B. Iriarte, Aleksa Obradović, Mine H. Wernsing, Lee E. Jackson, Botond Balogh, Jason A. Hong, M. Timur Momol, Jeffrey B. Jones, and Gary E. Vallad
  32. 32. Steps
  33. 33. Advantages  Fast processing ,low cost, high yield  Good at targeting and entering cells  Mostly target specific types of cells  Used as virus-induced gene silencing (VIGS) in reverse genetic studies 2003 Aug;30(4):296-303. Virus-induced gene silencing in plants. Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC.
  34. 34.  Express proteins in plants for the - Study of gene function - Production of vaccines - Study of metabolic engineering - Analysis of plant-microbe interactions Hum Vaccin. 2011 Mar;7(3):331-8. Epub 2011 Mar 1. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Chen Q, He J, Phoolcharoen W, Mason HS
  35. 35. Disadvantages  Worst effects to plants by –Producing severe disease –Giving undesired products –Affecting the plant adversely (due to highest mutation rate) http://plantsciences.utk.edu/stewart.htm
  36. 36. Cosmid  Derived from bacteriophage & plasmid  Cohesive sites + plasmid = cosmid  Less used for plant transformation  Carry DNA fragments of about 40 kb  E.g. US 8298819 B2 J Virol. 2000 Oct; 74(19): 8972–8979.PMCID: PMC102093 Generation of Mutant Murine Cytomegalovirus Strains from Overlapping Cosmid and Plasmid Clones Mariam E. Ehsani, Tshge W. Abraha ,Cecile Netherland- Snell,Niklaus Mueller, Meghan M. Taylor, and Barry Holwerda
  37. 37. Cohesive ends or sticky ends A single-stranded end of a linear duplex DNA molecule which can form hydrogen-bond with a complementary single-strand base sequence from the end of the same or another DNA molecule http://www.dnalc.org/view/15019-Cohesive-sticky-ends-and-their- significance-in-genetic-engineering-Paul-Berg.html

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