Upload
jeemcarlofagelapula
View
276
Download
2
Embed Size (px)
DESCRIPTION
Technical Course
Citation preview
Plant Tissue Culture
T.C. Refers to technique of growing
plant cells, tissues, organs, seedsor other plant parts in a sterile
environment on a nutrient medium
HistoryIn 1902 Haberlandt
proposed that single plant cells could be cultured
Haberlandtdid not culture them himself
1930’sWhite worked on T.C.discovery of plant growth
regulators
1930’simportance of vitamins was
determined for shoot and root culturing
1930’sIndole-Acetic AcidIAAdiscovered in 1937
IAA2,4-DDicambaNAAIBAall synthetic hormones
1957-58Miller and SkoogUniversity of Wisconsin -
Madisondiscovered Kinetin
Kinetina cytokininplays active role in
organogenesis
1958Steward developed somatic
embryo from carrot cells
1958-60Morel cultured orchids and
dahliasfreed them from a viral
disease
1962Murashige and Skoogpublished recipe for MS
Medium
60’s & 70’sMurashige cloned plants in
vitropromoted development of
commercial plant T.C. labs
1966raised haploid plants from
pollen grains
1972used protoplast fusion to
hybridize 2 species of tobacco into one plant
contained 4N
4Nall chromosomes of both
plants
70’s &80’sdevelop techniques to
introduce foreign DNA into plant cells
beginning of genetic engineering
T.C. Mediafunctionsprovide H2Oprovide mineral nutritional
needs
T.C. Mediaprovide growth regulatorsProvide vitaminsprovide organic compounds
T.C. Mediaprovide access to
atmosphere for gas exchangeserve as a dumping ground
for plant metabolites
T.C. MediaH2O is usually distilledminerals must provide 17
essential elementsenergy source and carbon
skeletons - sucrose is preferred
Vitaminsthiaminepyridoxinnicotinic acidbiotin
Vitaminscitric acidascorbic acidinositol
Growth Regulatorsauxins and cytokininsgibberellic acidabscissic acid
pH of mediausually 5.0-5.7
Mediamust be sterileautoclave at 250 F at 15 psi
for 15 minutes
T.C. StagesExplanting- Stage Iget plant material in sterile
culture so it survivesprovide with nutritional and
light needs for growth
Stage IIrapid multiplicationstabilized culturegoal for a commercial labdifficult and time consuming
to maintain
Stage IIoccurs in different pathways
in different plants
Rooting - Stage IIImay occur in Stage IIusually induced by changes in
hormonal environmentlower cytokinin concentration
and increase auxin
Rootingmay skip stage III and root
in a greenhouse
Stage IVtransplantation and aftercareusually done in greenhousekeep RH high (relative
humidity)
Stage IVgradually increase light
intensity and lower RH after rooting occurs
allows plants to harden and helps plants form cuticle
Cuticlewaxy substance promotes
development of stomatesplants in T.C. don’t have
cuticle
Explantportion of plant removed and used
for T.C.Important featuressizesource - some tissues are better
than others
Explantspecies dependentphysiological age - young
portions of plant are most successful
Explantdegree of contaminationexternal infestation - soak
plant in sodium hypochlorite solution
Explantinternal infection - isolate
cell that is not infectedroots - especially difficult
because of soil contact
Explantherbaceous plantssoft stemeasier to culture than woody
plants
Patterns of multiplication
stage II - light 100-300 foot candles
callus - shoots - rootsstage III - rooting - light
intensity 1000-3000 foot candles
Genetic transformation
permanent incorporation of new or foreigh DNA into genome of cell
Transformation methods
protoplast fusioncell wall is enzymatically
removed from cell
Protoplastsnaked plant cellsfrom 2 different plants can
be mixed together and forced to fuse
Protoplast fusionresults in heterokaryon cell containing two or more
nuclei from different cellshomokaryon - from same
cell
Protoplast fusionallowed to regenerate cell
wall and then grow into callus
callus turns to shoots
Shotgun approachDNA coated micro bullets of
gold or tungstonshot into growing cellsDuPont holds the patent
Shotgun approachinjures cellsrandom success rate
PEGPolyethylene glycolpores open similar to
electroporation
Ti PlasmidsTumor inducingAgrobacterium temefasciensinfect cells with
agrobacterium which contains desired DNA
Ti Plasmidsmonocots resist
agrobacterium infectionresearchers are working to
overcome this
Luciferasean enzyme put into tobacco using Ti
plasmid
Luciferasewhen transformed tobacco
plants are watered with solution containing Luciferin
they break it down and emit light
Luciferaseglowing in the darklike a fire fly
Screening techniquesused to identify if culture
has taken on desired new trait
Examplessensitivity to antibioticscolorsensitivity to excess
deficiencies of substances in growth media
Conventional plant breedingegg cell gives half the
chromosomes and almost all of the cytoplasm
male only gives its chromosomes
Cont…….This condition is called
maternal cytoplasmic inheritance
Microinjectionsingle cells from culture are
held stationary with gentle suction
injected with a tiny syringe loaded with DNA
Microinjectiondone under electron
microscope
Electroporationdesired DNA in solution
outside cellhigh energy pulses - 50,000
voltsfor a millisecond
Electroporationcause tiny pores to openallows DNA to enter the cell