Presentation on:

Plant transformation vectors and their types

Presented to:Dr. Hamid Manzoor

Presented by:Hina Mushtaq …………... BSBT-02Zahra Naz ……………….. BSBT-04

07th May, 2015

Institute of Molecular Biology & Biotechnology, Bahauddin Zakariya

University, Multan, Pakistan.

Contents

Plant transformation Vectors Types of vectors Plant transformation vectors Plasmids Viruses Bacteriophages

– Advantages – Disadvantages

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__%20Biotechn

ology%20-%20Plant%20Biotechnology.html

Steps of Plant

Transformation

Plant Transformation

Methods1.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.

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

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

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

In plants

Plasmids Viruses Bacteriophag

es Cosmids

Plant Physiol. 2007 Dec; 145(4): 1118–1128.doi: 10.1104/pp.107.106104PMCID: PMC2151730Focus Issue on Vector Systems for Plant Research and BiotechnologyDelivery of Multiple Transgenes to Plant CellsMery Dafny-Yelin and Tzvi Tzfira

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

Classification

Classification of plasmidso Fertility plasmid

e.g. F plasmid of E. colio Col plasmid

e.g. ColE1 of E. colio Resistance plasmid

e.g. RP4 in Pseudomonaso Degradative plasmid

e.g. TOL of P. putidao Virulence plasmid

e.g. Ti plasmids of A. tumefaciens

file:///C:/Users/%20/Documents/d.html

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

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

Advantages

Occur naturally in bacteriaHave different restriction sites.Replicate completely independent of bacteriaGenes are easily inserted into plasmidsEasily transformed into bacteria

https://answers.yahoo.com/question/index?qid=20061217124130AAsvKqk

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

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

Agrobacterium tumefaciens

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

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 example: pUC19Resistance to ampicilline.

Contains portion of the lacZ which codes for beta-galactosidase.

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/

Examples

1.Cauliflower mosaic virus based vectors.

2.Cowpea mosaic virus3.Bean pod mottle virus (BPMV)4.TMV based vectors.5.Potato virus X (PVX) 6.Bean yellow dwarf virus7.Bacteriophage Lambda Vectors 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613721/

Characteristics of viral vectors

Safety Low toxicity Stability Cell type specificity

http://www.ncbi.nlm.nih.gov/pubmed/12828943

Viruses are used in two ways

–Virus directly inserted into plant

–Virus indirectly inserted (bacteria)

http://www.ncbi.nlm.nih.gov/pubmed/12828943

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

Small insertions (10-30 bp) in various sites abolished infectivityThe largest insert is 256-531 bpCaMV genome can be inserted into Ti vector

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

Bacteriophage Lambda Vectors

Viruses that can infect bacteria 1000 times more efficient than

plasmid vectors Clone DNA fragments inrange of 10,000 - 20,000 bps

Bacteriophage. 2012 Oct 1; 2(4): 215–224.Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophagesTwo possible strategies for improving bacteriophage persistence for plant disease controlFanny 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

Steps

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.

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

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

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: PMC102093Generation of Mutant Murine Cytomegalovirus Strains from Overlapping Cosmid and Plasmid ClonesMariam E. Ehsani, Tshge W. Abraha ,Cecile Netherland-Snell,Niklaus Mueller, Meghan M. Taylor, and Barry Holwerda

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