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 ä Organogenesis ä Somatic 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 Somatic embryos Repetitive embryogenesis = cloning

Embryo growthPhysiological maturity Dry-down for storage - propagation Somatic embryogenesis

Germination and plant recovery

Part II: Getting DNA into plant cells Main methods ä Agrobacterium tumefaciens ä Biolistics [gene gun]

Agrobacterium is a natural plant genetic engineer

The Ti-plasmid is required for crown gall disease T-DNA = Transferred DNA Ti = Tumor inducing

The Ti Plasmid Hormones cause gall growth, opines are special nitrogen sources

Agrobacterium transfer is complex Borders 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 E Export out of the cell

Opine Synthesis Cytokinin Synthesis Auxin Synthesis Border Antibiotic Resistance Gene of Interest Reporter Gene Cut and replace Disarming the T-DNA

A chimeric gene n Level of expression n Constitutive n Tissue-specific n Polyadenylation site n Provides stability to mRNA Coding sequence Promoter Terminator l 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 Engineered plant cell

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 biolistics GUS 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 culture stimulate division from plant cells

Antibiotics in plant tissue culture limit growth to engineered cells Other 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 plants Number 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 methods Expert view on chance of unintended consequences for food quality National Research Council (2004)

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 LSAGLAGLSAGLAG

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?