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

Part I:

Getting whole plants back from cultured cells

OrganogenesisSomatic embryogenesis

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

Shoots usually are produced first, then roots in organogenesis

Somatic embryogenesis – shoot-root axis differentiated as a unit

Immature cotyledon Somaticembryos

Repetitive embryogenesis = cloning

Embryo growth Physiologicalmaturity

Dry-down for storage - propagation

Somatic embryogenesis

Somatic embryogenesis

Germination and plant recovery

Part II:

Getting DNA into plant cellsMain methodsAgrobacterium tumefaciensBiolistics [gene gun]

Agrobacterium is a natural plant genetic engineer

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

Cocultivation of Agrobacterium with wounded plant tissues

Agrobacterium in contact with wounded plant tissues during cocultivation

The gene gun

Plastic bullet DNA on gold particles

.22 caliber charge

Stopping plate

Firing pin

Gene gun bombardment of plant tissues in Petri dish

DNA coated metal particles after “gene-gun” insertion into tissues

Transgenic cassava via biolisticsGUS reporter gene gives blue color

Part III:

Selection of transgenic cells

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)

Hormones in plant tissue culturestimulate division from plant cells

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)

Antibiotic selection of transgenic tissues in poplar

Summary of steps in Agrobacterium transformation

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 structure and orientation can vary Single, simple copies much preferred for stability

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?