Plant Tissue Culture Matt Jakubik. T.C. * Refers to technique of growing plant cells, tissues,...
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Transcript of Plant Tissue Culture Matt Jakubik. T.C. * Refers to technique of growing plant cells, tissues,...
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Plant Tissue CultureMatt Jakubik
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T.C. Refers to technique of growing
plant cells, tissues, organs, seedsor other plant parts in a sterile
environment on a nutrient medium
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HistoryIn 1902 Haberlandt
proposed that single plant cells could be cultured
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Haberlandtdid not culture them himself
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1930’sWhite worked on T.C.discovery of plant growth
regulators
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1930’simportance of vitamins was
determined for shoot and root culturing
A, D, E, K, C, and B Complex
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1930’sIndole-Acetic AcidIAAdiscovered in 1937
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IAA2,4-DDicambaNAAIBAall synthetic hormones
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1957-58Miller and SkoogUniversity of Wisconsin -
Madisondiscovered Kinetin
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Kinetina cytokininplays active role in
organogenesis
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1958Steward developed somatic
embryo from carrot cells
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1958-60Morel cultured orchids and
dahliasfreed them from a viral
disease
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1962Murashige and Skoogpublished recipe for MS
Medium
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60’s & 70’sMurashige cloned plants in
vitropromoted development of
commercial plant T.C. labs
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1966raised haploid plants from
pollen grains
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1972used protoplast fusion to
hybridize 2 species of tobacco into one plant
contained 4Nall chromosomes of both plants
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70’s &80’sdevelop techniques to
introduce foreign DNA into plant cells
beginning of genetic engineering
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T.C. Mediafunctionsprovide H2Oprovide mineral nutritional
needs
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T.C. Mediaprovide growth regulatorsProvide vitaminsprovide organic compounds
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T.C. Mediaprovide access to
atmosphere for gas exchangeserve as a dumping ground
for plant metabolites
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T.C. MediaH2O is usually distilledminerals must provide 17
essential elementsenergy source and carbon
skeletons - sucrose is preferred
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Vitaminsthiaminepyridoxinnicotinic acidbiotin
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Vitaminscitric acidascorbic acidinositol
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Growth Regulatorsauxins and cytokininsgibberellic acidabscissic acid
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pH of mediausually 5.0-5.7
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Mediamust be sterileautoclave at 250 F at 15 psi
for 15 minutes
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T.C. StagesExplanting- Stage Iget plant material in sterile
culture so it survivesprovide with nutritional and
light needs for growth
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Stage IIrapid multiplicationstabilized culturegoal for a commercial labdifficult and time consuming
to maintain
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Stage IIoccurs in different pathways
in different plants
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Rooting - Stage IIImay occur in Stage IIusually induced by changes in
hormonal environmentlower cytokinin concentration
and increase auxin
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Rootingmay skip stage III and root
in a greenhouse
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Stage IVtransplantation and aftercareusually done in greenhousekeep RH high (relative
humidity)
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Stage IVgradually increase light
intensity and lower RH after rooting occurs
allows plants to harden and helps plants form cuticle
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Cuticlewaxy substance promotes
development of stomatesplants in T.C. don’t have
cuticle
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Explantportion of plant removed and used
for T.C.Important featuressizesource - some tissues are better
than others
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Explantspecies dependentphysiological age - young
portions of plant are most successful
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Explantdegree of contaminationexternal infestation - soak
plant in sodium hypochlorite solution
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Explantinternal infection - isolate
cell that is not infectedroots - especially difficult
because of soil contact
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Explantherbaceous plantssoft stemeasier to culture than woody
plants
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Patterns of multiplication
stage II - light 100-300 foot candles
callus - shoots - rootsstage III - rooting - light
intensity 1000-3000 foot candles
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Genetic transformation
permanent incorporation of new or foreign DNA into genome of cell
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Transformation methods
protoplast fusioncell wall is removed by
enzymes from cell
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Protoplastsnaked plant cellsfrom 2 different plants can
be mixed together and forced to fuse
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Protoplast fusionresults in heterokaryon cell containing two or more
nuclei from different cellshomokaryon - from same
cell
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Protoplast fusionallowed to regenerate cell
wall and then grow into callus
callus turns to shoots
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Shotgun approachDNA coated micro bullets of
gold or tungstonshot into growing cellsDuPont holds the patent
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Shotgun approachinjures cellsrandom success rate
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PEGPolyethylene glycolpores open similar to
electroporation
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Ti PlasmidsTumor inducingAgrobacterium temefasciensinfect cells with
agrobacterium which contains desired DNA
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Ti Plasmidsmonocots resist
agrobacterium infectionresearchers are working to
overcome this
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Luciferasean enzyme put into tobacco using Ti
plasmid
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Luciferasewhen transformed tobacco
plants are watered with solution containing Luciferin
they break it down and emit light
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Luciferaseglowing in the darklike a fire fly
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Screening techniquesused to identify if culture
has taken on desired new trait
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Examplessensitivity to antibioticscolorsensitivity to excess
deficiencies of substances in growth media
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Conventional plant breedingegg cell gives half the
chromosomes and almost all of the cytoplasm
male only gives its chromosomes
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Cont…….This condition is called
maternal cytoplasmic inheritance
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Microinjectionsingle cells from culture are
held stationary with gentle suction
injected with a tiny syringe loaded with DNA
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Microinjectiondone under electron
microscope
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Electroporationdesired DNA in solution
outside cellhigh energy pulses - 50,000
voltsfor a millisecond
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Electroporationcause tiny pores to openallows DNA to enter the cell