DR MUHAMMAD ARSHAD Institute of Environmental Science & Engineering, NUST, Islamabad

1. DR MUHAMMAD ARSHAD Institute of Environmental Science & Engineering, NUST, Islamabad Genetic engineering for the environment: concepts and applications Ecolab NIBGE NUST

2. Contents • Introduction • Steps in genetic engineering • Genetic transformation of scented Pelargonium: a case study • Environmental applications • Biosafety concerns • Conclusions & Perspectives

3. Introduction Genetic Engineering  technology involved in removing, modifying, or adding genes to a DNA • Genetic Engineering term: Jack Williamson (1951) in “Dragon's Island” • DNA's role in heredity (Hershey and Chase, 1952) and DNA structure (Watson and Crick, 1953)

4. Steps in genetic engineering Gene isolation Vector construction Genetic transformation Selection of GMO Genetic engineering

5. Pb Gene isolation Metabolomics Proteomics ↑or ↓ expression of genes as compared to control Different metabolites -Pb +Pb Organic acids Enzymes Synthesis pathway Transformation Gene function Candidate genes

6. pMDC162 (Curtis & Grossniklaus, 2003) LB RB Vector construction LB = left border RB = Right border hpt = Hygromycin phosphotransferase p35S, NOS TER = Promoters Uid A = GUS gene

7. Transformation Transformation • Agrobacterium mediated • Biolistic (gene gun) • Electroporation • Microinjection Selection Regeneration Transformation Transfer & integration of a foreign sequence.

8. Genetic transformation of scented Pelargonium A case study

9. Regeneration • Development of a plant from a cell. • Toti-potency Culture of explants on medium Initiation of shoots 18 weeks Developed shoots

10. Regeneration Cytokinins 6-Benzylaminopurine (BAP), Kinetin (K), Zeatin (Z). Explant Morphogenesis/ Embryogenesis Morphogenesis Auxins Naphthalene Acetic Acid (NAA), Indole-Acetic Acid (IAA). Callus (Yemets et al., 2003) > 30 combinations were tested. Rhizogenesis (Saxena et al. 2007) Embryogenesis (Murthy et al., 1999) Thidiazuron 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ)

11. Regeneration Strategy 1: Assays containing NAA, BAP, Kinetin & Zeatin Regeneration efficiency BAP NAA Zeatin No. of shoots per explant  Low mg L-1 Regeneration Efficiency (%) = No. of explants forming shoots× 100 Total explants

12. Regeneration Strategy 2: MS + TDZ 2 weeks MS or MS + BAP/NAA Regeneration efficiency (%) • No. of shoots/explant No. of responding explants BAP NAA mg L-1

13. Regeneration Strategy 3: MS + BAP/NAA + TDZ 10 µM 2 weeks Regeneration efficiency (%) MS + BAP/NAA BAP NAA mg L-1 93% for both cultivars with 100 shoots par explant  To be tested for transformation

14. pMDC162 (Curtis & Grossniklaus, 2003) LB RB Vector Strain of Agrobacterium containing a disarmed Plasmid pTIBo542 (S.B. Gelvin, Purdue University, USA)

15. Inoculation = bacteria + explants Un-inoculated control + Antibiotics Un-inoculated control without Antibiotics Inoculated + Antibiotics Selection Genetic Transformation Selective Marker  Reporter gene (GUS)  RB LB Vir Disarmed Ti Plasmid Regeneration

16. Transformation Inoculation 10-30 min Renewal of media  4 weeks Co-culture 3 days 17 weeks Pre-culture 2 weeks Culture 6 weeks GUS, PCR, Southern Blot Observations Molecular Analyses Rooting 8 weeks Observations

17. Transformation GUS test Histo-chemical test  Coloration in the presence of X-Gluc Atomic cultivar, strain C58 Leaf fragments Leaf fragment (×20) Root hairs (×100) Root (×20) Root (×30)

18. Transformation PCR screening Polymerase Chain Reaction 1 2 3 4 5 6 7 8 9 10 1200 bp PCR+ plants of Atomic cultivar, after inoculation with C58 containing pMDC162:2X35S GUS vector.

19. Transformation After inoculation with C58 AttarAtomic Total explants 117 104 Regenerated shoots 394 1106 Rooted plantlets 4 107 GUS+ 4 82 PCR+ 2 20 Transformation efficiency 0.5% 1.8%  No. of PCR+ plants × 100 Number of regenerated shoots on selection medium Conclusion  potential transformants were obtained.

20. Environmental applications

21. Environmental applications Crop plants with improved characteristics Insect/pest resistant varieties e.g. Bt Cotton Efficient to utilize inputs/ nutrients Use of insecticides/ pesticides inputs required Environment

22. Genetically engineered bacteria Production of enzymes/hormones for accelerated degradation of pollutants (Liu et al. 2008) • Genetically engineered fungi Production of chemicals, pharmaceuticals and enzymes: environment friendly (Meyer, 2008) Environmental applications • Genetically engineered algae Biomarker for quality of water

23. Genetically engineered virus Lithium ion battery by using virus (Belcher-MIT-, 2009) • Biofuel production Modifying lignin for enhanced biofuel production (Simmons et al. 2010):  Greenhouse gases Environmental applications

24. Phytoextraction Hyperaccumulation Biomass >1000 mg Pb kg-1 DW (Baker et al., 2000) > 3 t ha-1 y-1 (Schnoor, 1997) Rapid growth Extracted Quantity QPb=[Pb]× Biomass Environmental applications • Phytoremediation Engineering for desired characteristics

25. Biosafety concerns Tolerance to pesticides Allergenicity Gene flow Fate of transgene in the environment GM plants Nutritional composition Antibiotic resistance Influence on non-target species Digestibility

26. Conclusions & perspectives Very promising technique « Broad application » Biosafety issues; To be addressed Potential for food security & environmental protection Life cycle assessment; For GM plants

27. Thanks for your attention.