Plant transformation Introduction of individual gene(s) of interest into plant genome Genetic modification with or without integration May include regeneration step Expected phenotypes to be analyzed (lab/field) Stable inheritance in the offspring

Plant transformation Position effect from random integration of transgene differential gene expression endogenous gene transgene different phenotypes from a transformation

Target gene DNA insert from the same or different species Designed to work under controlled conditions require specific promoter for activation of gene expression

Gene expression control Under the control of promoter Target gene to express all the time / certain stage / in response to signal whole plant / some tissues Weak / Strong promoter determine amount of gp

Genetic marker Detection / Selection of transformed plant Marker gene: bacteria or plant origin Often linked to the transgene (same cassette) Confer a phenotype to transformed cell/tissue Selectable / Screenable marker

Selectable marker Antibiotic / Herbicide resistance gene Transformed plants survive / grow better under selection pressure For selection / segregation ratio based on ability to grow on selective agent

Screenable marker GUS / LUX / CAT / lacZ genes (gp=enzyme) Transformed plants screened for phenotype by histochemical staining fluorimetric assay For selection / gene expression study (level / pattern)

Transformant selection

Plant transformation Transient transformation easy, rapid and convenient approach for transient assay no integration / similar to bacterial plasmid Stable transformation integration of foreign DNA in host genome stably maintained after cell division

Transient transformation Gene expression assay in protoplast eg. study of functional domain of promoter deleted promoter:marker transformation with fusion constructs phenotypic analysis

Transient transformation Leaf protoplast: most common explant developmental stages vary different response / expression Protoplast transformation electroporation polyvalent cation: PEG or PLO

Transient transformation Electroporation electric shock to open the membrane DNA uptake / membrane seal Polyvalent cation activate DNA uptake by endocytosis

Electroporation

Electroporator&Cuvette

Endocytosis

Stable transformation Stable integration of transgene into plant chromosome Transgene / genetic marker replication and function in plant cells Transgenic plant by regeneration transgene inherited and active

Stable transformation DNA-mediated gene transfer polyvalent cation / electroporation / microinjection Integration during replication Homologous recombination of plant gene/foreign DNA Protoplast as explant Difficult for cereal / legume

Microinjection

Stable transformation Particle gun mediated gene transfer to overcome limitation of protoplast regeneration more developed explants eg. embryogenic tissue DNA travel through several cell layers DNA-coated particles penetrating by force

Stable transformation Particle gun mediated gene transfer gold or tungsten microparticles a.k.a. gene gun or biolistics used with all plant species

Stable transformation Agrobacterium mediated gene transfer reliable and versatile method neoplasia-causing soil bacteria: Agrobacterium natural ability to transfer DNA into plant

Stable transformation Agrobacterium mediated gene transfer bacterial plasmid as transforming vector A. tumefaciens: Ti plasmid crown gall disease A. rhizogenes: Ri plasmid hairy root disease

Agrobacterium infection

Agrobacterium T-DNA Agrobacterium mediated gene transfer plasmid DNA to be transferred: T-DNA flanked by 25-bp imperfect direct repeats left border and right border mediating proteins: Vir ABCDEG

Agrobacterium Ti-plasmid

Agrobacterium T-DNA Natural T-DNA genes Changes in differentiation and development of transformed plant cells Neoplastic phenotype by disrupting hormone balance Ti: increase auxin / cytokinin synthesis Ri: alter hormone response induce root differentiation

Agrobacterium T-DNA Natural T-DNA genes Enzymes for opines synthesis and secretion nopaline / octopine / agrocinopine amino acid / sugar derivatives as a source of carbon and nitrogen

Natural DNA transfer process Wounded plant cells release phenolic compounds acetosyringone: to trigger following steps chemotaxis of bacteria to plant cells binding of bacteria to plant cell wall induction of virulence/vir gene expression

Natural DNA transfer process Vir A: constitutively expressed transmembrane sensing protein In response to phenolic compound Autophosphorylation of Vir A Phosphorylated Vir A to activate Vir G protein Chemotaxis of bacteria

Natural DNA transfer process Phosphorylated Vir G as transcriptional activator of remaining Vir loci Vir C: to bind a sequence at right border Vir D2: strand-specific ss endonuclease to initiate nick at bottom strand

Natural DNA transfer process VirE2: ssDNA-binding protein to protect T-strand from nucleases Vir B as bacterial membrane protein to transfer DNA from bacteria to plant cell

Natural DNA transfer process Other chromosomal genes att, chvA, chvB and pscA for tight binding of bacteria to plant cells cel: formation of cellulose fibrils for bacterial aggregation

Agrobacterium for Transformation Recognition site for DNA transfer direct repeats on T-DNA border T-DNA genes: no role in DNA transfer/integration removal with no effect Vir genes: function in trans fashion

Agrobacterium for Transformation Modification of Ti plasmid binary vector cointegrative vector

Binary vector Split Ti plasmid to 2 molecules separate transgene and vir genes Subclone target DNA in a binary vector MCR / selectable marker / RB / LB ori active both in E. coli and Agrobacterium Transgene to replicate in E. coli and Agrobacterium

Binary vector Vir genes already on a T-DNA-deleted Ti plasmid for expression in Agrobacterium E. coli culture to check construct and multiplication Agrobacterium culture for plant transformation

Cointegrative vector Deletion derivative of Ti plasmid Replacing T-DNA with defined DNA sequence Require helper or intermediate vector for subcloning of DNA insert

Cointegrative vector Intermediate vector MCR for insert cloning Selectable markers Sequence homologous to defined sequence in cointegrative vector Replication (ori) only in E. coli

Cointegrative vector Transform Agrobacterium with intermediate vector DNA transfer from intermediate vector to cointegrative vector by homologous recombination Then Agrobacterium culture for plant transformation

Gene transfer method With transgene-containing Agrobacterium Protoplast cocultivation Tissue explant inoculation Seed imbibition In planta transformation

Agrobacterium transformation

Explant inoculation

Regeneration of transgenics

Application of transgenic plants To define specific sequence (promoter) for directing differential expression To express foreign genes and determine or analyze phenotypes of interest To engineer crop plants for useful agronomic traits

Examples of transgenic crops Most well known transgenics Roundup Ready: glyphosate tolerant crops Bt crops: Cry toxin of Bacillus thuringiensis Other traits: etc.

Herbicide tolerant plant

Bt & BT toxin

BT plant

Safety assessment of transgenics Evaluation prior to release Environmental safety / Food safety Information on transgenic development GM plant information (plant and product) Safety assessment / Regulatory decision Regulatory approval & management

Safety assessment of transgenics Criteria Host organism / Donor organism Modification process Molecular characterization Genetic stability of introduced trait Expressed material

Safety assessment of transgenics Criteria Gene transfer to related plant Horizontal gene transfer Weediness potential Secondary and non-target traits Insect resistance management Others