Plant Tissue Culture -generation of whole plants from a single cell or tissue-

1. Plant Tissue Culture-generation of whole plants from a single cell or tissue- Biotech II

2. Plasticity and Totipotency • Plasticity: ability of plants to survive varying conditions by altering their metabolism, growth and development • Ability to regenerate lost parts • Ability to begin cell division from tissue of the plant • Ability to generate any tissue or organ from any other type of cell • Totipotency: maintenance of genetic potential of all cells (any cell can be stimulated to become any other type of cell)

3. The Culture Environment • In vitro (vs. In vivo) • Meets both chemical and physical needs • Many plant cultures are not photosynthetic and thus require sugar (usually sucrose)

4. Plant Cell Culture Media • 3 Basic components: • Essential elements (supplied as salt mixtures) • Vitamins and amino acids • Sugar The Essential Elements: Macroelements: N, P, K, Mg, Ca, S, C Microelements: Mn, I, Cu, Co, B, Mo, Fe, Zn

5. Plant Growth Regulators (hormones) • Auxins-promote cell division and cell growth, root formation and buds (Indoleacetic acid-IAA) • Cytokinins-promote cell division, shoots, and buds • Gibberellins-promotes seed and leaf bud germination • Abscisic acid (ABA)-inhibits cell division • Ethylene-promotes ripening

6. Culture Steps • Obtain meristem cells aseptically • provide the hormones to induce Callus formation-auxin and cytokinin • At callus stage, gene of interest can be introduced. • Lastly, modified callus tissue is aseptically transferred to differentiation media to induce shoot and root formation.

7. Isolate plant cells Transform gene into plant Grow undifferentiated callus Transform cells Select cells Grow transgenic plant Redifferentiate callus

8. Methods to introduce genes into Plants • Ti plasmid and Agrobacterimtumefaciens • Protoplast fusion (cellulase removal of cell wall and combination with a protoplast from another plant) • Direct uptake (not efficient) • Microinjection • Electroporation • Particle bombardment (gene gun) • Liposomes –small lipid spheres filled with plasmid • Engineered chloroplasts-chloroplast DNA not put into pollen, so no problem with unintended mixing with wild plants

9. Why Genetically Modify Plants? • Enhanced Taste, Nutrition, and/or Color • Survival (climate and pesticides)-Bt gene • Shelf life • Vaccines • Phytoremediation • Future fuel?

10. Crops • 60% of US crops are GM • 60-70% of these are transformed with the BT gene • Major GM crops: corn, cotton, and soy

11. Detection of Genetically Modified Foods

12. Foods in US that may contain GM products… • US FDA approval in place for: • Corn • Soy • Papaya • Canola • Potato • Chicory • Rice • Squash • Sugarbeet • Tomatoes • Note: approval does not necessarily mean these crops are being distributed • Database of GM crops: www.agbios.com

13. Bacillus thuringiensis Clone the gene into Ti plasmid Delta endotoxin crystal Bt gene ori Ti plasmid Ti genes

14. GO STOP • Engineer the gene • so that it will be expressed: • Promotor for transcription initiation • Stop sequence for transcription termination Bt gene ori Ti plasmid Ti genes Antibiotic resistance

15. Testing for GMO’s: Two General Approaches ELISA: Tests for presence of proteins expressed from genetic modifications Pro: Quick, cheap, low tech Con: Crop specific, protein instability PCR: Tests for presence of inserted foreign DNA Pro: ID many different GM crops, DNA stability Con: Expensive, more time consuming

16. Testing for GMO’s Using PCR…. • General Steps: • Grind food • Extract DNA from sample • Test sample DNA for viable plant DNA (PCR) • Test sample DNA for genetic modifications (PCR) • Visualize results via electrophoresis

17. PCR Reaction Components • Template - the DNA to be amplified • Primers - 2 short specific pieces of DNA whose sequence flanksthe target sequence • Forward • Reverse • Note: primers are what select the specific sequences to be amplified • Nucleotides - dATP, dCTP, dGTP, dTTP • Taq DNA polymerase – thermophillic enzyme from hot springs • Magnesium chloride - enzyme cofactor • Buffer - maintains pH & contains salt

18. The PCR Reaction: Review • How it Works: • Heat (94oC) to denature DNA strands • Cool (59oC) to anneal primers to template • Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA • Repeat for 40 cycles http://www.phgfoundation.org/tutorials/dna/4.html

19. NOS Terminator sequence CaMV35s promotor sequence Inserted gene PRIMER PCR-based detection of GM specific sequences…. PRIMER PCR Amplification 200 bp GMO-specific fragment GMO specific primers detect CaMV 35S and NOS sequences: used as transcription initiation (RNA-P binding) and terminator sequences for most genes inserted into Ti vectors

20. Detection of plant sequences: universal primer • Confirms that viable plant DNA was extracted. • Uses highly conserved chloroplast gene from Photosystem II – part of the light reaction of photosynthesis. • Primers are designed to amplify this gene • Amplification produces a 455 bp fragment

21. Sample Results: 1: non-GMO food with plant primers 1 2 3 4 5 6 7 2: non-GMO food with GMO primers GMO positive 3: Test food with plant primers 4: Test food with GMO primers 5: GMO positive template with plant primers 6: GMO positive template with GMO primers 7: PCR MW Ruler 1 2 3 4 5 6 7 GMO negative

22. Trouble shooting • False Positives • Contamination-sterile technique; 10% bleach to clean pipette barrels, mortars & pestles, bench tops; barrier tips for all steps to correct. • False Negatives • No DNA extracted • Possible food type or possibly primers do not work on that plant species • InstaGene matrix transferred to PCR reactions