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

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

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

1.   Auxins-promote cell division and cell growth, root
     formation and buds (Indoleacetic acid-IAA)
2.   Cytokinins-promote cell division, shoots, and buds
3.   Gibberellins-promotes seed and leaf bud
     germination
4.   Abscisic acid (ABA)-inhibits cell division
5.   Ethylene-promotes ripening
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.
                     Isolate plant cells

Transform
gene into                                  Grow
                                           undifferentiated
plant                                      callus




                                       Transform cells




            Select cells

                                                     Grow
                                                     transgenic
                             Redifferentiate
                                                     plant
                             callus
Methods to introduce genes into Plants
     Ti plasmid and Agrobacterim tumefaciens
     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
Why Genetically Modify Plants?
 Enhanced Taste, Nutrition, and/or Color
 Survival (climate and pesticides)-Bt gene
 Shelf life
 Vaccines
 Phytoremediation
 Future fuel?
Crops
 60% of US crops are GM
 60-70% of these are transformed with the BT
  gene
 Major GM crops: corn, cotton, and soy
Detection of Genetically Modified
             Foods
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
               Bacillus thuringiensis
Clone the
gene into Ti
plasmid                        Delta endotoxin crystal




                         Bt gene

                                                     ori
                          Ti plasmid

                         Ti genes
Engineer the gene             GO                 STOP

so that it will be
expressed:
  •Promotor for
  transcription initiation
  •Stop sequence for
  transcription termination        Bt gene


                                                          ori
                                    Ti plasmid

                                   Ti genes
                                                        Antibiotic
                                                        resistance
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
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
             • Template - the DNA to be amplified
             • Primers - 2 short specific pieces of DNA whose
               sequence flanks the target sequence
                  èForward
PCR               çReverse
Reaction          Note: primers are what select the specific
                   sequences to be amplified
Components   • Nucleotides - dATP, dCTP, dGTP, dTTP
             • Taq DNA polymerase – thermophillic enzyme
               from hot springs
             • Magnesium chloride - enzyme cofactor
             • Buffer - maintains pH & contains salt
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
                                                                      NOS
                                         CaMV35s                    Terminator
                                         promotor   Inserted gene   sequence
                                         sequence
                                                                                 PRIMER

PCR-based                       PRIMER
detection of GM
specific                                                      PCR Amplification

sequences….

                                          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
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
Sample Results:
1: non-GMO food with plant primers
                                                     1   2   3   4   5   6   7
2: non-GMO food with GMO primers

3: Test food with plant primers       GMO positive
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
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

				
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