Genetic Engineering Mechanics

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Genetic Engineering Mechanics Zhanyuan Zhang,
Ph.D. Plant Transformation Core
Facility University of Missouri-Columbia
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Genetic Engineering Process

• Cell and Tissue Culture as a Tool
• Gene Delivery Systems
• Vector Construction
• Transformation and Transgenic Recovery
• Transgene Analysis
• Application of Genetic Engineering
• Regulations of GMO

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Plant Tissue Culture As a Tool

• Cell and Tissue culture ABC
• Regeneration potential and totipotency
• In vitro tissue culture conditions (coupling art
  with science)
• Regeneration systems
• Organogenesis (direct and indirect
  organogenesis)
• Somatic embryogenesis
• Cell suspension culture
• Protoplast culture
• Anther culture

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In Vitro Culture Response
Root or callus induction
Auxin concentrations
Shoot formation
Cytokinin Concentrations
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Plant In Vitro Tissue Culture Conditions

• Plant growth regulators
• Cytokinin Zeatin, kinetin, BAP, TDZ, 2iP
• Auxin IAA, IBA, 2,4-D, GA3
• Types of plant tissue culture media
• Murashige Skoog (1962) MS
• Gamborg et al. (1968) B5
• Chu (1975) N6
• Linsmaier and Skoog (1964) LS
• Gelling agents
• Agar, Gellan gums or Gelrite
• Equipment and culture vessels

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B5 Medium
Macronutrients (Major salts) (mg/L) KNO3,
Potassium nitrate 2500 CaCl2H2O, Calcium
chloride 150 MgSO47H2O, Magnesium sulfate
250 (NH4)2SO4, Ammonium sulfate
134 NaH2PO4H2O, Sodium phosphate 150 Micron
utrients (Minor salts) H3BO3, Boric acid
3 MnSO4H2O, Manganous sulfate 10 ZnSO47H2O,
Zinc sulfate 2 KI, Potassium iodide
0.75 Na2MoO42H2O, Sodium molybdate
0.25 CuSO45H2O, Cupric sulfate
0.025 CoCl26H2O, Cobalt chloride
0.025 Vitamins Myo-inositol
100 Nicotinic acid 1 Pyridoxine-HCl
1 Thiamine-HCl 10 Ion-EDTA FeSO47H
2O, Ferrous sulfate 28 NaEDTA,
Ethylenediamine teraacticacid, disodium salt
37.5

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Equipment and Culture Vessels
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Organogenesis
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Somatic Embryogenesis
(Courtesy from Wayne Parrott Lab)
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Cell Suspension Culture
(Liu et al., 1998)
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Protoplast Culture
(Courtesy from Levee et al., unpublished)
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Anther Culture
(1n)
(Courtesy from Smith and Belzer, 2002)
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Gene Delivery Systems

• Agrobacterium-mediated T-DNA transfer
• Tissue culture-based approach
• Non-tissue culture approach
• Direct DNA delivery
• Biolistic-mediated DNA delivery (Gene-gun)
• Electroporation
• Direct DNA injection

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Crown-Galls Tumors tumefaciens
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T-DNA Transfer From Agrobacterium To Plant Cell
T-DNA
Wild Type
RB
RB
Plant cell
LB
Chromosome
Ti-plasmid
T-DNA
Disarmed
RB
LB
Chromosome
Ti-plasmid
Binary vector
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VirA
VirA
VirA
VirA
Wounded sites
Bacterial chromosome
chvA chvB chvF
VirG
VirG
Ti-plasmid
VirD2
VirE
VirE
virB, virC, virD, virE1, virE2, virF, virH
Vir region
VirE
VirE
VirE
LB
RB
VirE
RB
LB
VirD1
Binary vector
VirE
VirE
VirE
VirE
B4
B10
B9
B1
VirD2
VirD2
B11
VirE
VirE
VirE
VirE
B6
B7
VirE
VirE
VirE
VirE
VirD2
VirE
VirE
VirE
VirE
Cytoplastic receptors
Agrobacterium tumefaciens
Plant Cell
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Gene-gun Biolistic PDS 1000/He
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Vector Construction
MCS
RB
Binary Vector
Expression Cassette
LB
T-DNA
GOI
Promoter
Terminator
Cloning Vector
MCS
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Plant Transformation and Recovery
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Non-Tissue Culture Approach Flower Dipping
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Transgene Analysis Genomic Southern Blot
W-13-12
W-14-20
W-14-24
W-14-29
W-11-42
W-13-28
W-13-24
W-14-8
Kb 23.0 9.4 4.4
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Application of Genetic Engineering

• Metabolic engineering
• Functional genomics
• Stress (biotic and abiotic) tolerance
• Phytoremediation
• Vaccine production

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Regulations of GMO

• Containment (BL1-P, BL2-P, , by NIH)
• Disposal (biohazards GMO reproductive tissues)
• Movement (cross state lab to field)
• Field tests (application protection zones)
• GMO Labeling