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