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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 Part I: Getting whole plants back from cultured cells Organogenesis Somatic embryogenesis Organogenesis – sequential differentiation of new plant organs (shoots, roots) First step is dedifferentiation into callus after treatment with the plant hormone auxin Leaf-discs 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 Somatic embryogenesis Embryo growth Physiological maturity Dry-down for storage propagation Somatic embryogenesis Germination and plant recovery Part II: Getting DNA into plant cells Main methods Agrobacterium tumefaciens Biolistics [gene gun] Agrobacterium is a natural plant genetic engineer The Ti-plasmid is required for crown gall disease T-DNA = Transferred DNA T-DNA Ti plasmid Ti = Tumor inducing The Ti Plasmid Hormones cause gall growth, opines are special nitrogen sources Opine Catabolism Cytokinin Synthesis Auxin Synthesis T-DNA Right Border Left Border 200000 bp Virulence region independent of TDNA Opine Metabolism Agrobacterium transfer is complex Borders define start and end of T-DNA Left Border Right Border Nick by VirD2 Nick by VirD2 New strand synthesis Strand displacement Preparation of T-strand Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Export out of the cell Disarming the T-DNA Border Border Auxin Synthesis Cytokinin Synthesis Opine Synthesis Cut and replace Antibiotic Resistance Gene of Interest Reporter Gene A chimeric gene Promoter Coding sequence Level of expression Constitutive Tissue-specific Terminator Polyadenylation site Provides stability to mRNA 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 T-DNA Ti Plasmid Engineered plant cell Agrobacterium tumefaciens 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 Firing pin .22 caliber charge Stopping plate 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 DNA inserted gene flanking 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 Over 2000 crop varieties derived from mutagenesis have been commercialized. Calrose 76 semi-dwarf rice High oleic sunflower Rio Red grapefruit Comparing GE to other breeding methods Expert 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 LAG LSAG Intron LSAG LAG 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?