PLANT GENETIC ENGINEERING

Amit Kumar SahooII M.Sc. Biosciences15151

Amit Kumar SahooII Msc B15151

Ti plasmid derived vector systems,protocols for transformation

Aum Sri Sai Ram

INTRODUCTION

WHAT ARE PLASMIDS ?

-PLASMIDS are double stranded, closed circular DNA molecules, which exist in the cell as extra chromosomal units.

-Transmitted from one bacterium to another (even of another species) via horizontal gene transfer.

- Engineered plasmids are widely used as vectors in DNA cloning .

Transgenic plants – An introductionTransformation – the process of obtaining transgenic plants.

Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome.

Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between

Agrobacterium and plants, which resulted in the development of methods to alter the bacterium

into an efficient delivery system for genetic engineering in plants.

Most common genes (and traits) in transgenic or biotech cropsherbicide resistanceInsecticide resistanceBt genes in field corn (maize)virus-resistance (coat-protein) genes

Plant Transformation Methods

Physical Chemical BiologicalMicroinjectionPressureBiolistics - gene gun/particle bombardmentElectroporationSilica/carbon fibersLazer mediated SAT

PEGDEAE-dextranCalcium phos-phateArtificial lipidsProteinsDendrimers

A. TumefaciensA. RhizogenesVirus-mediated

Agrobacterium tumefaciens “The Natural Genetic Engineer”

• A. tumefaciens is a gram-negative soil

bacterium which naturally transforms plant

cells, resulting in crown gall (cancer)

tumors.

• Infects plants through breaks or wounds.

• Tumor formation is the result of integration

of T-DNA (Transfer DNA) in plant genome.

-A. tumefaciens causes crown gall disease, A. rhizogenes causes hairy root disease, and A. rubi causes cane gall disease.

-Curing a particular plasmid and replacing this plasmid with another type of tumorigenic plas-mid can alter disease symptoms.

-Perhaps a more meaningful classification system divides the genus Agrobacterium into “bio-vars” based on growth and metabolic characteristics.

-As a genus, Agrobacterium can transfer DNA to a remarkably broad group of organisms includ-ing numerous dicot and monocot angiosperm species.

-In addition, Agrobacterium can transform fungi,including yeasts,ascomycetes,and basid-iomycetes.Recently,Agrobacterium was reported to transfer DNA to human cells.

-Several virulence (vir) loci on the Ti plasmid, including virC (367, 368) and virF (220, 267), were shown to determine the range of plant species that could be transformed to yield crown gall tumors.

-However, it is now clear that host range is a much more complex process, which is under the genetic control of multiple factors within both the bacterium and the plant host.

Some Points to remember

Ti- Plasmid

About

• A Ti or tumour inducing plasmid(approx.size 200 kb each) is a plasmid that often, but not al-

ways, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium

rhizogenes use to transduce its genetic material to plants.

• The Ti plasmid is lost when Agrobacterium is grown above 28 °C. 

• The plasmid has 196 genes that code for 195 proteins. There is one structural RNA. The

plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding

genes. There are no pseudogenes.

• The modification of this plasmid is very important in the creation of transgenic plants.

• Genes in the virulence region are grouped into the operons virABCDEFG, which code for the

enzymes responsible for mediating conjugative transfer of T-DNA to plant cells.

Ti plasmid and virulence genesThe Ti plasmid has three important region:-

1.T-DNA region: This region has the genes for the biosynthesis of auxin (aux),cytokinin (cyt) and opine

(ocs), and is flanked by left and right borders.

T-DNA borders- A set of 24 kb sequences present on either side (right & left) of T-DNA are also

transferred to the plant cells.

It is clearly established that the right border is more critical for T-DNA transfer.

2. Virulence region: The genes responsible for the transfer of T-DNA into host plant are located outside

T-DNA and the region is reffered to as vir or virulence region .

At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir C1, vir D1, D2,

vir D4 and vir E1,E2.

3. Opine catabolism region: This region codes for proteins involved in the uptake and metabolisms of

opines.

Besides the above three there is ori region that responsible for origin of DNA replication which permit the

Ti plasmid to be stably maintain in A. tumefaciens.

Ti Plasmid stucture

Infection Process

Cellular process of Agrobacterium–host interaction

Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129

Ti Plasmid-Derived Vector Systems Using Ti plasmid as a vector it is possible to

insert a desired DNA sequence (gene) into the T

DNA region of Ti plasmid.

There are several limitations to use Ti plasmids

directly as cloning vectors :-

LARGE SIZE.

TUMOR INDUCTION PROPERTY.

ABSENCE OF UNIQUE RESTRICTION SITES.

• Agrobacterium plasmids are disarmed by

deleting naturally occurring T-DNA encoded

oncogenes and replacing them with foreign

genes of interest.

The right and left border sequences of T-DNA

which is required for T-DNA integration.

A multiple cloning site.

An origin of replication

A selectable marker gene

Though Ti plasmids are effective natural vectors they had certain limitations.

• The phytohormone produced by transformed cells growing in culture prevents their regen-eration into mature plants. Hence auxins and cytokinin genes must be removed from the Ti –plasmid derived cloning vector.

• The opine synthesis gene must be removed as it may divert plant resources into opine pro-duction in transgenic plant.

• Generally, Ti- plasmids are large in size (200-800kb)For effective cloning, large segments of DNA that are not essential for cloning has to be removed.

• As Ti plasmid does not replicate in E.coli Ti-plasmid based vectors require an ori that can be used in

E.coli.

To overcome these constraints, Ti plasmid based vec-tors were organized with the following components:

• A selectable marker gene that confers resistance to trans-formed plant

cells. As these marker genes are prokaryotic origin, it is nec-essary to put them under the eukaryotic control (plant) of post transcrip-tional regulation signals, including promoter and a termina-tion- poly adenylation sequence, to ensure that it is efficiently expressed in transformed plant cells. • An origin of replication that allows the plasmid to replicate

in E.coli. • The right border sequence of the T-DNA which is necessary

for T-DNA integration into plant cell DNA. • A polylinker (MCS) to facilitate the insertion of cloned gene

into the region between T-DNA border sequences. • As these cloning vectors so organized lacked vir genes,

they cannot effect the transfer and integration of T-DNA region into host plant cell. So two Ti-plasmid derived vector systems were developed. They include:

• 1. Binary vector system • 2. Co-integrate vector system

Binary vector

t-DNAVIR genesPlasmid DNA

BacterialChromosomeBacterial ORI

Ampicillin resistance

Construction of vector with disired genes

Making of Co-Integrate Vectors-In this strategy, both the T-DNA with our gene of interest and vir region are present in the same vector used for transformation.

-At first; an intermediate vector is made using E.coli plasmid + vir region + T-DNA borders + origin of replication+pBR 322 sequences.

-Second vector is a disarmed pTi vector = gene of interest+ some markers+pBR322 sequences.

-Both intermediate vector and disarmed pTi has some sequences in common (pBR322 sequences).-Therefore by homologous recombination, co-integration of two plasmids will take place within Agrobacterium.

Now we have a co-integrate vector that has both T-DNA with our gene of in-terest with in the T-DNA borders and vir region. This complete vector is used for transformation eg:pGV2260.

Binary vector strategy: Two vector strategyHere two vectors are used. This vector was devised based on the knowledge that vir region need not be in the same plasmid along with T-DNA for T DNA transfer.

Binary vector consists of a pair of plasmids1) A disarmed Ti plsmid: This plasmid has T-DNA with gene of interest + ori for

both E.coli and Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: Bin 19

2) Helper Ti plasmid has virulence region that mediates transfer of T-DNA in mi-cro Ti plasmid to the plant.

In general, the transformation procedure is as follows:the recombinant small replicon is transferred via bacterial conjugation or direct transfer to A. tumefaciens harboring a helper Ti plasmid, the plant cells are co-cultivated with the Agrobacterium, to allow transfer of recombinant T-DNA into the plant genome, and transformed plant cells are selected under appropriate conditions.1.binary vector system involves only the transfer of a binary plasmid to Agrobacterium without any integration.

2.This is in contrast to co-integrate vector system wherein the intermediate vector is transferred and integrated with disarmed Ti plasmid.

3.Due to convenience, binary vectors are more frequently used than co-integrate vectors.

Compared with co-integrated vectors, binary vectors present some advantages:-No recombination process takes place between the molecules involved.-Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used, which increases transfer efficiency from E. coli  to Agrobacterium.This vector system is most widely used nowadays. Different types of binary vectors have been devised to suit different needs in a plant transformation process.

Agrobacterium mediated transformationThe important requirements for Agrobacterium-

mediated gene transfer in higher plants are as follows:-

The plant explants must produce acetosyringone for activation of Vir genes.

The induced Agrobacterium should have access to cells that are competent for transformation.

Explants include cotyledon, leaf, thin tissue layer, peduncle, hypocotyls, stem, microspores

Process of Transformation

Transformation ProtocolsTransformation was performed using mi-nor modifications of published protocols, using a.Leaf disk b.Scutellum-derived callus, or c.Floral dip methods, respectively.

An ExampleExample-scutellum-derived callus method

Transformation Protocol for Rice – Abbreviated

Seed plating on 2N6 – dark ↓ 4 weeksSubculture onto 2N6 – dark ↓ 4 – 10 daysCo cultivation onto 2N6-AS – dark ↓ 3-7 daysSelection on 2N6-TCH – dark ↓ 4 weeks, subculture onto 2N6-TCH every 2 weeksTransfer proliferating calli onto 2N6-TCH-dark ↓ 2 weeksRegeneration onto RGH6-dark ↓ 7 daysTransfer to light ↓ 4-6 weeksPlantlets onto ½ MSH - light ↓Transfer plants to the glasshouse

Seed Material: Oryza sativa L. ssp japonica cvs. Millin or Nipponbare.Steps-1. Callus Induction2. Callus Subculture3. Bacteria Preparation4. Transformation5. Callus washing6. Selection7. Regeneration8. Plantlet Formation-To ensure that the gene transfer did not result from contamination with Agrobacterium cells, controls including species specific PCR, selective plating, and use of a tagged binary vector were implemented.

-Thus, diverse plant associated bacteria, when harbouring a disarmed Ti plasmid and binary vector(or presumably a cointegrate or whole Ti plas-mid), are readily able to transfer TDNA to plants. The Ti plasmid is self transmissable, perhaps indicating the existence of a ubiquitous natural mechanism effecting horizontal gene transfer from bacteria to plants.

Procedure for plant transformation

IMAGE: Mol bio of the cell by Albert (pg no:599)

Regeneration, Selection And Detection... Regeneration: for shoot organogenesis, cytokinin (lower

amounts of auxin) are required Selection: two antibiotics are required

• An antibiotic to kill the Agrobacterium, while not affecting the plant's cell growth and division• a second antibiotic allows growth of transformed shoots (w/selectable marker) but inhibits growth of untransformed plant cells.

Detection of the "trait" gene PCR methods can detect the presence of the "trait" DNA protein detection methods are used where a gene product is produced that defines the trait verification of the incorporation of the trait gene into the plant's chromosome:

• by Southern hybridization• by demonstrating transfer of the trait to the original transformant's progeny.

 Ø Scientists can insert any gene they want into the plasmid in place of the tumor causing genes and subsequently into the plant cell genome.  Ø By varying experimental materials, culture conditions, bacterial strains, etc. scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn. Ø This method of gene transfer enables large DNA strands to be transferred into the plant cell without risk of rearrangement whereas other methods like the Gene Gun have trouble doing this .

Ø The vast majority of approved genetically engineered agriculture has been transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer.

Ø Original problems existed in that Agrobacterium tumefaciens only affects dicotyledonous plants. Ø Monocotyledon plants are not very susceptible to the bacterial infection.

Benefits and Problems with Agrobacte-ria

References:1.www.asgct.org › General Public › Educational Re-sources2.www.link.springer.com

3.www.bios.net

4.en.wikipedia.org

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