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Science and GMO-relevant technology
• Genes and genomes – last week– Genomes and their inheritance and variation– Genes and their structure– Important methods: Gene cloning, PCR and
microarrays
• Biotechnology - today– Basic concepts of cloning/regeneration– Transformation methods– Transgene structure/expression
Organogenesis – sequential differentiation of new plant organs (shoots, roots)
Leaf-discs
First step is de-differentiation into callus after treatment with the plant hormone auxin
Somatic embryogenesis – shoot-root axis differentiated as a unit
Immature cotyledon Somaticembryos
Repetitive embryogenesis = cloning
The Ti-plasmid is required for crown gall disease
Ti plasmid
T-DNA
T-DNA = Transferred DNA
Ti = Tumor inducing
The Ti PlasmidHormones cause gall growth, opines are special nitrogen sources
T-DNA
Opine Catabolism
Right Border
Opine MetabolismVirulence region independent of T-DNA
Left Border
Auxin Synthesis
Cytokinin Synthesis
200000 bp
Agrobacterium transfer is complexBorders define start and end of T-DNA
Right Border
Nick by VirD2
Left Border
Nick by VirD2
Strand displacement
New strand synthesis
Preparation of T-strand
Vir EVir EVir EVir EVir EVir EVir EVir E Vir EVir EVir EVir E
Vir EVir EVir EVir EVir EVir EVir EVir E Vir EVir EVir EVir E
Export out of the cell
OpineSynthesis
CytokininSynthesis
AuxinSynthesis
BorderBorder
AntibioticResistance
Gene ofInterest
ReporterGene
Cut and replace
Disarming the T-DNA
A chimeric gene
Level of expression Constitutive Tissue-specific
Polyadenylation site Provides stability to mRNA
Coding sequencePromoter Terminator
Mix and match parts
Example of a map of plasmid used in plant transformation
GUS gene encodes glucuronidase (cleaves pigment to make blue color): GUS reporter gene enables easy visualization of successful transformation, and where and when genes are expressed
Agrobacterium engineering
Gene of interest
Agrobacterium tumefaciens
Engineeredplant cell
T-DNA
Ti Plasmid
Only a few cells get engineered
Challenge: Recover plants from that one cell so new plant is not chimeric (i.e., not genetically variable within the organism)
Antibiotics in plant tissue culturelimit growth to engineered cellsOther kinds of genes can also be used to favor transgenic cells (e.g., sugar uptake, herbicide resistance)
Analysis of transgenic plantsNumber of gene copies can vary
Junction fragment analysis reveals number of gene insertion sites
Restriction enzyme sites shown with arrows
flanking DNA inserted gene flanking DNA
Transgene expression level varies widely between insertions (“events”)Partly due to failure to control where gene inserts in genome
Interpreting significance of GE’s unintended effects on genome
• Lots of unintended genetic change in breeding
• Lots of genetic variation in gene content and organization
• No urgency to regulate traditional breeding
Varieties derived from induced mutations
Calrose 76 semi-dwarf rice
High oleic sunflower
Over 2000 crop varieties derived from mutagenesis have been commercialized.
Rio Red grapefruit
Comparing GE to other breeding methodsExpert view on chance of unintended consequences for food quality
National Research Council (2004) http://books.nap.edu/execsumm_pdf/10977.pdf
Extensive natural genetic diversity in gene structure/content (maize)Natural deletions of genes/chromosome sections
Summary of some GE biological issues to consider
• Events = unique gene insertion– They vary widely in level/pattern of expression due to
chromosomal context / modification during insertion– The unit of regulatory consideration at present– Mutagenic changes at insertion site highly variable (deletions,
duplications)– Can be “read-through” (Agro DNA beyond T-DNA transferred)
• Stability of gene expression and gene silencing – A large number of insertions are not expressed– Some lose/change expression over time– Must select and test events carefully – single copy preferred
Summary of some GE biological issues to consider
• Somaclonal variation = unintended mutagenesis due to tissue culture & regeneration system– Can be substantial, varies widely depending on culture system– Must weed out via crossing, intense selection of events
• Increasing use of RNAi (RNA interference), as a general means of gene suppression in research and commerce– A way to knock out specific genes, inhibit viruses– Genes with inverted repeat DNA create double-stranded RNA,
which induces sequence-specific RNA degradation or inhibition of translation – very active area of basic and applied research
Intron LSAG LAGLSAGLAG
Discussion questions• What aspects of gene transfer are most unclear?
– What are most important to understand for interpreting biotechnologies?
• Should individual gene transfer events be the focus of safety evaluations? – Or should the type of gene in a specific crop be
regulated instead? • Should GE crops that modify the expression of
native kinds of genes (ie, not introduce novel kinds of genes) be regulated at all?