Dr. James Saunders                                          April 7, 1997
Lecture Outline                                         Protoplast Lecture

I - Protoplasts Produced

A. Mechanical
     1 - cut off wall
     2 - slicer - random cut-off wall

B. Enzyme (5.5. - 6.0 pH, sucrose osmoticum) 
     buffer, cellulase

     4 hours digest cell wall - obtain membrane-bound cell - (like animal)

     Apolar proplasts - chloroplasts
          even distribution, have large vacuoles - repository 
               Garbage dump bag

     Found nictotine stored in vacuole - contains defense compounds - insect
	attack, natural insecticide

     Lower sugar concentration-cells burst releasing vacuole

     Density gradient centrifuge to obtain purified vacuolar preparation

     Intact vacuoles


A. Herbivore - (Larva - Army Worm larva stores nicotine and serves as chemical
	protection against parasitoid wasps)

B. Cell fusion - hybrids (of plants that are NOT sexually compatible)
     Promoted by:
     1 - Solutions - such as PEG
     2 - Electrical charge used to damage membrane - come back together as
		single membrane; however, the cells must be in close contact so that low
		voltage electrical current can be used to charge cells (Dielectroporesis).

C. Maintain cell viability
     1 - Cell wall reforms
     2 - Hanging drop culture used to form small callus

     Formed new plant
     Fusion: all genes or lose some genes

D. Sheep
     Took out nucleus of egg and put in new somatic nucleus - shocked
     Put back into mother - used egg to provide cloning environment

E. Video - Colorado Potato Beetle
     Beetle tolerant to pesticides
     Leptine in plants associated with insect resistance
     Somatic mating to produce potato plants high in leptine which resists
	herbivory by Colorado Potato Beetles

F. Applications
     Contaminated soil - high heavy metals such as zinc.
     Small number of plants will grow in high zinc
     remediation of soil - plants
          Concentrated in leaves, leaves harvested and then recover zinc in
		smelter - no longer a disposal problem.
          Used protoplast fusion between small zinc accumulating plant and
		larger plant Brassica
          Produced larger plant which can accumulate zinc

     Can extend studies - use genes which are associated with capacity to
	accumulate metals - DNA specific for metal uptake

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