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•6/1/2018
•1
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