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Prof. Fahd M. Nasr

Faculty of SciencesLebanese University

Beirut, Lebanon

https://yeastwonderfulworld.wordpress.com/

Biol328 - B3212Molecular

BiotechnologyLectures 19 and 20

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Plant Biotechnology

Plant BiotechnologyTraditional

crossbreedingRecombinant

DNA techniquesHeterosis

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The results of traditional Agbiotech are not that bad, but they are time-consuming

teosinte

cob

corn

Wild tomato (Lycopersicon pimpinellifolium)

D= 1 cm

Results of ‘Natural’ Crop Sex• Domestication of crops• Improved crop traits for

enhanced yield and/or grain quality

• Genetic modification of crop species by – Rearrangement of genes within a species via natural or

directed-pollination– Incorporation of genes from "wild" or "close" relatives

via natural cross-pollination

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Early domestication: cow being milked in ancient Egypt

Artificial Selection among "Mustards" Brassica oleracea

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Limits to Crop Breeding?• Slow process to develop new varieties

– Not uncommon for 8 – 10 years from start to finish using traditional breeding techniques

• Desired new or improved traits not always present in crop species– Disease or insect resistance– Physiological improvements– Stress tolerance– Grain or plant composition

Transgenic Example: Bt

• Bt genes originate from the soil bacteria, Bacillus thuringiensis– Researchers have identified and isolated the Bt

genes– Introduced into corn DNA– Bt genes that successfully incorporate into corn

DNA express the insecticidal Bt protein in corn plant grown by farmer

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Bt plants

Genetic engineering: plants

• Considerable agricultural importance• Considerable controversy regarding

health and environmental safety–Long-term effects are unknown

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Making a transgenic plant requires several steps

• You need to have your gene of interest (YFG) cloned into an expression vector

• The actual transformation– Introduction of YFG into the plant tissue

• Several techniques are available• Regeneration of the transformed tissue

growing a new plant from transformed cells• Selection of transformants• Analysis of transgene expression

Transformation techniques

• Plant transformation how plant work and improve crop plant characteristics

• Stable insertion of transgene genome

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Transformation

• Transformation refers to the generation of transgenic organisms, i.e. the introduction of a gene of choice into the genome of an organism

• Referred to as genetic engineering

• 2 major methods for transformation–Ti plasmid from Agrobacterium

tumefaciensAgrobacterium-mediated gene transfer

–Direct gene transfer methodsGene gun to inject DNA-coated

micropellets into cells (biolistics)

Genetic engineering: plants

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The biology of Agrobacterium

• Soil-borne, Gram-negative, rod-shaped, mobile bacterium rhizosphere

• Plant biologists and biotechnologists• Causative agent of "crown gall" disease

economically important (grapes, apples, ..)• A pathogen of dicots ability to transfer

genes into the plant genome• Problems with monocots particle

bombardment

Ti plasmid and crown gall disease• A portion of the Ti plasmid is inserted

into the plant chromosome• This system can be harnessed to create a

useful mechanism for transforming plants

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Injured plantBacteria

incorporatedTumorForms

Molecules sensed by the

Agrobacterium(chemotaxis)

Agrobacterium

Agrobacterium cell Plant cell

Ri-plasmid

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Agrobacterium-mediated gene transfer

• The ability to transfer genes unique feature of inter-kingdom gene transfer

• For biotechnologist plant transformation method

Genetic engineering of plants with Agrobacterium tumefaciens

• A. tumefaciens used for genetic engineering

• Contains T-DNA (bacterial plasmid Ti)

• Genes could be integrated into the plant chromosomes when the T-DNA is transferred Tumor induced by A. tumefaciens

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A.Tumefaciens gall is not a tiny thing

Ti Plasmid

Tumor-producing genes

Virulence region

Opine catabolism

ORI

T-DNAregion

DNA between L and R borders istransferred to plantas ssDNA

T-DNA encoded genes can be substituted by target genes

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Crown-gall disease• Depends on Ti plasmid• T-DNA region is transferred integrates

the genome– Genes for hormone biosynthesis– Genes for plant metabolites (opines and

agropines) biosynthesis• A + C gall• Plant metabolites source of carbon and

energy

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Arginine + Pyruvate

Arginine + a-ketoglutarate

The Ti plasmid• T-DNA transfer functions are encoded in a

specific part of the plasmid (virulence genes)• Transfer occurs by a mechanism almost identical

to bacterial conjugation• Insert a gene into the T-DNA let the mechanism

of DNA transfer take over transfer into plant cells• Ti plasmids are too large to manipulate so a

methodology to insert DNA into the T-DNA has been developed

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Sexual conjugation

Agrobacterium mediated transformation

• Use of this naturally occurring gene transfer process as a transformation tool– Ti-plasmid could be disarmed the tumor

inducing genes on the T-DNA are eliminated– Replace the undesirable genes with YFG!

• The modern expression vectors can replicate in both E. coli and A. tumefaciens– Construct the plasmid in E. coli– Harvest large quantities transfer it to A.

tumefaciens via electroporation

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Ti-plasmid based vectors

Binary systemsCo-integrated vectors

Binary cloning plasmidwith gene of interest(no vir genes)

Disarmed Ti plasmidcapable for infectionIntermediate vectorwith T-region and gene of interest (transferred by conjugation)

Form co-integrated plasmid after homologous recombination on T-DNA

Helper vectorfor infection(with vir genes)

First approach: binary system

• Binary cloning vector (disarmed Ti)– E. coli and A. tumefaciens ori of Rep– Or single broad-host-range Ori of Rep– No vir genes

• Cloning steps in E. coli A. tumefaciens• Recipient strain

– Helper plasmid– With complete set of vir genes– Lacking T-DNA region

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First approach: binary system

• Helper plasmid vir gene products transfer functions mobilize T-DNA from binary vector

• T-DNA transfer is initiated from RB– Insert PSM next to LB– Few binary vectors contain two PSMs

LB and RB

Binary cloning vector plasmid

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Binary systems: two plasmids

Virulence region

T DNA region removed

ori for A. tumefaciens

Gene of interest

Plant selectable marker

Bacterial selectable marker

ori for A. tumefaciensori for E.coli

HELPER plasmidDisarmed

Ti plasmid

DISADVANTAGE: plasmids with two different origins of replication may be unstable in E. coliADVANTAGE: small vectors are used, which increases transfer efficiency from E. coli to AgrobacteriumNo intermolecular recombination is needed

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Procedure for creation a transgenic plant

1. Both plasmids are transfected into A. tumefaciens

2. Plant cell culture is infected with A. tumefaciens

3. Products of vir genes mobilize T-DNA (gene of interest)and transfer it to plant chromosome

Polylinker Kan-resistance geneT-DNA Repeat T-DNA Repeat

Gene of interest

4. Plant cells are selected on kanamycin5. Presence of transgene confirmed by PCR

6. Whole plant could be grown from transformed cells

Second approach: co-integrate vectors

• Cloning co-integrate vector– PSM, Target gene, RB and no vir genes– E. coli Ori of Rep and BSM

• Recombination with– Disarmed Ti-plasmid lacking RB and oncogenes with A. tumefaciens Ori of Rep and vir genes

– To generate recombinant Ti plasmid

• Cloning vector can be maintained as cointegrate

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Second approach

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Production of transgenic plants by use of co-integrated Ti plasmids

Co-integrated vectors hybrid Ti-plasmids

Long homologies required between the co-integrate and Ti plasmid difficult to engineer and use

Relatively inefficient gene transfer compared to the binary vectors

Right now rarely used

DISADVANTAGES

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• Typical plant expression vectors– E. coli origin of replication– A BSM (bacterial antibiotic resistance gene)– A plant promoter (CaMV 35S promoter or

other inducible or tissue specific promoters)– A plant selectable marker (PSM) herbicide

or antibiotic resistance gene (often kanamycin)

How to make a transgenic plant?

How to make a transgenic plant?• For Agrobacterium vectors

– A separate origin of replication– (May be) a separate antibiotic resistance gene

• After construction– The vectors are typically introduced in E. coli

and then propagated– The plasmid is then harvested and is either used

for direct transformation or is introduced in A. tumefaciens

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• Why make transgenic plants?–Improvement of crop plants by

introduction of new genes–Promoter studies–Find out gene function

overexpression or silencing

Transformation

Agrobacterium mediated transformation

• A. tumefaciens and A. rhizogenes are pathogenic soil bacteria– Contain a Ti (tumor inducing) or Ri (root

inducing) plasmid

– A small piece (the T-DNA is transferred and integrates into the plant genome)

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Agrobacterium mediated transformation

• The T-DNA– flanked by a LB and a RB (25 bp direct repeats)– LB and RB recognized by virD1 and virD2

endonucleases– virD2 nicks the border sequence and binds to

the 5’ end– T-DNA unwinds and is transferred as a ssDNA– T-DNA is coated with virE2 (NLS) plant’s

nucleus integration into the genome

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L Border and R Border

Transformation process Infecting the plant tissue with A. tumefaciens

carrying the newly designed Ti-plasmidThe general method involves Incubate a leaf cut in a suspension of bacteriaThe cells on the edge of the punch are transformed Selects for transformants (based on PSM)Kills Agrobacterium (carbenicillin) Stimulates shoots (high cytokinin:auxin ratio)The shoots are placed on a root-inducing medium (low

C:A ratio)The resulting plantlets are eventually transferred to soil

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The end