1. Plant Genetic Engineering (Plant Transformation) Nono Carsono, PhD. Dr. rer. nat Suseno Amien Anas, PhD.

2. Genetic transformation/modification of plant? Introduction of exogenous DNA into a plant cell - transient: no incorporation of exogenous DNA (transgenes) into the genome - stable: incorporation into genome Transgene - the genetically engineered gene added to a (plant) species Product - Transgenic plant: plant containing transgenes introduced by genetic engineering/modification/ transformation (not classical breeding) Transformation of multicellular organisms: - can not directly transform every cell - transformation involves one cell which then regenerates an entire organism Transient expression of GFP Stable expression of GFP

3. Why use Genetic Transformation? Accelerate the breeding process – Introduce/enhance desired trait in an established genetic background Extend the gene pool (with care, especially if potential for entry into the food chain) – Select genes from any Kingdom (with care, especially if potential for entry into the food chain)

4. Diff. between conv. breeding and genetic engineering ParameterConventional Breeding Genetic engineering LevelWhole plantCell/organelle PreciseMany genesOne gene or some Taxonomy barrierWithin species/genusNo restriction CertaintyGenetic change- hard to estimate Genetic change quite easy to estimate

5. Bt Corn Reduces: Insecticide Mycotoxin Application Production

6. What are the Uses of GM Plants? Research – Largest number of transgenic plants are currently created for research purposes Knock-outs, over-expression, modified proteins K. Yamaguchi-Shinozaki, JIRCAS, Japan stress-inducible promoter driving drought- and cold-responsive transcription factor wild type

7. What are uses of GM Plants? Commercial Applications – Altered agronomic traits Disease/insect resistance Virus resistance Herbicide resistance Salt/drought tolerance Cold tolerance Enhanced yields, other quantitative traits Phytoremediation Production of vaccine Application of Roundup herbicide Field following application time 2007

8. Other uses of GM Plants? Bioreactors / Molecular farming – Therapeutic proteins Human lactoferrin to treat iron deficiencies Antibodies – Vaccine production Antigen expression – HepC, HIV Dow AgroSciences Achieves World’s First Registration for Plant-Made Vaccines Indianapolis, IN - January 31, 2006 Dow AgroSciences LLC, a wholly owned subsidiary of The Dow Chemical Company, (NYSE: DOW), announced today that it has received the world's first regulatory approval for a plant-made vaccine from the United States Department of Agriculture (USDA) Center for Veterinary Biologics. This approval represents an innovative milestone for the company and the industry...

9. Other uses of GM Plants? Functional foods (humans and livestock) – Today: Golden rice Vitamin A enriched – Future directions: Boosted antioxidants Elevated content of specific minerals Removal of food allergens, carcinogens Greater public acceptance when the technology is shown to more greatly benefit consumers?

10. Golden Rice Scientists from Swiss and German universities have engineered two genes from daffodil and one bacterial gene into rice to produce provitamin A. GGPP Phytoene Lycopene beta-Carotene = provitamin A Phytoene synthase (psy) Phytoene desaturase (crtl) Lycopene ß-cyclase (lcy) (daffodil) (bacteria) Provitamin A biosynthesis pathway Funding: Rockefeller Foundation, Swiss Federal Institute Of Technology, European Community Biotech Program

11. An Overview of the Genetic Engineering/modification cycle

13. Prerequisites for genetic transformation Available gene of interest (incl. selectable marker and other regulatory sequences)- isolation and construct design Efficient method for genetic transformation- gene transfer Efficient method for regeneration (whole plant)- plant regeneration Promoter Termination sequence Bt coding sequence How the direction of transcription? Right or left? ?

14. CODING SEQUENCE INTRON poly A signal PROMOTER Building the Transgenes Plant Transgene bacterial genes antibiotic marker replication origin Plant Selectable Marker Gene Plasmid DNA Construct ON/OFF Switch Makes Proteinstop sign

15. Transformation Cassettes Contains 1. Gene of interest The coding region and its controlling elements 2. Selectable marker Distinguishes transformed/untransformed plants 3. Insertion sequences Aids Agrobacterium insertion

16. Transformation Steps Prepare tissue for transformation Introduce DNA Culture plant tissue Develop shoots Root the shoots Field test the plants Leaf, germinating seed, immature embryos Tissue must be capable of developing into normal plants Agrobacterium or gene gun Multiple sites, multiple years

17. Delivering the gene to the plant genome: Gene transfer methods Direct transfer of DNA - PEG-polyethylene glycol - Electrophoration Transfer of DNA via carrier - Microinjection - Particle bombardment Transfer of DNA via vector - Agrobacterium tumefaciens - Viral vector

18. Biolistic bombardment (gene gun) Transformation of Agrobacterium Cloned Gene in Vector DNA Molecule Protoplast transformation followed by cell wall regeneration Agrobacterium-mediated transformation of plant cell Migration and integration of gene into nucleus Plant cells grown in tissue culture Regeneration of genetically modified plant from tissue culture

20. Biolistic / Gene Gun

21. Plasmid vector Vector cut with EcoRI Donor DNA Donor DNA cut with EcoRI Donor DNA fragments Add DNA ligase Introduce into E. coli Tetracycline-resistant Bacterial colony from transformed cell Transformed cell Plasmids Recombinant DNA Selectable antibiotic resistance marker

24. The Next Test Is The Field Non-transgenics Transgenic plant Herbicide Resistance

25. Example: Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After

26. Roadmap Bioengineering Plants for the Future 1997200520152025 Efficient agriculture -Bt technology -Herbicide resistance etc Health food and quality -Amino acids -Oil -Starch Plant protection -Viruses -Nematodes -Fungi -Insects Plant production platforms -Vitamines -Fatty acids -Enzymes -Bio-polymers -Pigments -Pharmaceutical products -Fibers Stress resistance -Cold -Drought -Salinization

27. Nature Biotechnology 25: 271 (2007)

30. Transgenic Confirmation (Genotypic analysis) PCR for rapid screening Southern blot for precise gene detection Northern blot for transcription analysis Western blot for translation analysis, together with Ab-binding or enzymatic analysis Mendelian analysis for insertion locus and linkage analysis In situ hybridization for precise insertion locus analysis DNA methylation analysis for silencing potential analysis

31. Regulatory principles of transgenic crops: 1.Scientifically based, based on information of organism, used technology and effects to humans and environment 2.Product-based approach, use existing product-based legislation 3.Familiarity and substantial equivalence, experience with the use of that species. The determination is based on scientific literature and practical experience with the plant and similar plant varieties. 4. Case-by case, allow the development of knowledge that could inform criteria and requirement over time.

32. Regulatory principles: 5.Step-wise fashion, products should be assessed throughout the chain of development : From laboratory to greenhouse and finally large-scale field trial 6.Transparency 7.Precautionary principle/approach, derived from Rio Declaration, regulatory groups can make decisions about products based on scientific uncertainty. 8.Harmonization, sharing of or acceptance of another group ’ s review

33. Questions Do not forget..next week student presentation!