Techniques of MicropropagationTechniques of Micropropagation

Chapter 18Chapter 18

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

• I.) Axillary shoot formationI.) Axillary shoot formation– Meristem tip cultureMeristem tip culture

• Results in plantlets free from viruses, fungi and bacteria Results in plantlets free from viruses, fungi and bacteria (esp. when coupled with heat treatment)(esp. when coupled with heat treatment)

• Important for many herbaceous crops (carnations, Important for many herbaceous crops (carnations, mums, orchids, geraniums, banana, potato, sweetpotato)mums, orchids, geraniums, banana, potato, sweetpotato)

• With woody plants, meristems are often graftedWith woody plants, meristems are often grafted

– Axillary shoot cultureAxillary shoot culture• Reliably reproduces the genotype of the parent plant Reliably reproduces the genotype of the parent plant

(expands existing buds)(expands existing buds)

Carnation meristemCarnation meristem

Nodal shoot production at cotyledonary stageNodal shoot production at cotyledonary stage

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

• Adventitious shoot formationAdventitious shoot formation– Initiated directly on the explant or indirectly from callusInitiated directly on the explant or indirectly from callus– Results in high rates of multiplicationResults in high rates of multiplication– Results in increased aberrant (“off-type”) plantsResults in increased aberrant (“off-type”) plants– Parts used:Parts used:

• Leaf pieces (ie: African violet)Leaf pieces (ie: African violet)• Cotyledons (ie: conifers)Cotyledons (ie: conifers)• Immature inflorescence (ie: Hosta and daylily)Immature inflorescence (ie: Hosta and daylily)• Bulb scales (ie: Easter lily, hyacinths, etc.)Bulb scales (ie: Easter lily, hyacinths, etc.)

Bulblet formation in tissue cultureBulblet formation in tissue culture

Hosta cultureHosta culture

Hosta cultureHosta culture

Hosta cultureHosta culture

Types of micropropagationTypes of micropropagation

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

• III.) Callus, cell & protoplast culture systemsIII.) Callus, cell & protoplast culture systems– Can be subcultured and maintained indefinitelyCan be subcultured and maintained indefinitely

– Callus cultureCallus culture• Produced in response to wounding & hormonesProduced in response to wounding & hormones• Almost all plant parts can be induced to produce callusAlmost all plant parts can be induced to produce callus• Both auxins & cytokinins must be in the mediumBoth auxins & cytokinins must be in the medium• Can be induced to form organs (Organogenesis). Can be induced to form organs (Organogenesis).

Parenchyma produces meristems (= meristmoids)Parenchyma produces meristems (= meristmoids)• First done with tobacco & carrotFirst done with tobacco & carrot

Direct shoot production (organogenesis)Direct shoot production (organogenesis)

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

– Cell suspensionsCell suspensions• Produced from “friable” callus (= loose)Produced from “friable” callus (= loose)

• Maintained in shaker cultures or bioreactorsMaintained in shaker cultures or bioreactors

– Protoplast cultureProtoplast culture• Cell culture without cell walls (cellulase added to degrades the Cell culture without cell walls (cellulase added to degrades the

cell wall)cell wall)

• Only plasmamembrane remainsOnly plasmamembrane remains

• Osmotic pressure must be maintained to keep cells from Osmotic pressure must be maintained to keep cells from rupturing (mannitol used)rupturing (mannitol used)

• Why done? Secondary plant products that leak from the Why done? Secondary plant products that leak from the protoplasts are collected (ex: taxol, sanguinaria)protoplasts are collected (ex: taxol, sanguinaria)

Cell cultures on a shakerCell cultures on a shaker

Bioreactors for cells or protoplastsBioreactors for cells or protoplasts

Protoplast cultureProtoplast culture

Protoplast cultureProtoplast culture

Sanguinaria canadensisSanguinaria canadensis“bloodroot”“bloodroot”

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

• IV.) Somatic embryogenesis & Synthetic IV.) Somatic embryogenesis & Synthetic seedseed– Development of embryos without a zygote Development of embryos without a zygote

(i.e. from non-gamete cells)(i.e. from non-gamete cells)

– Roots and shoots develop simultaneously to Roots and shoots develop simultaneously to form embryoids (i.e.: carrots)form embryoids (i.e.: carrots)

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

– Arise from:Arise from:• Adventitious somatic embryogenesis (directly from Adventitious somatic embryogenesis (directly from

cells = embryogenic cells). Usually arise near zygotic cells = embryogenic cells). Usually arise near zygotic cellscells

• Induced somatic embryogenesis. Callus must form Induced somatic embryogenesis. Callus must form first (often in suspension culture). Usually conditioned first (often in suspension culture). Usually conditioned on high levels of auxin (2,4-D)on high levels of auxin (2,4-D)

– Uses:Uses:• Clonal propagationClonal propagation

• Genetic manipulation -using Genetic manipulation -using Agrobacterium Agrobacterium tumefascienstumefasciens or a gene-gun or a gene-gun

Somatic embryogenesis (soybean)Somatic embryogenesis (soybean)

Somatic embryogenesis (soybean)Somatic embryogenesis (soybean)

Somatic embryogenesis (sitka spruce)Somatic embryogenesis (sitka spruce)

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

• Environmental conditions during tissue Environmental conditions during tissue cultureculture– TemperatureTemperature

• 68 - 81°F68 - 81°F

• Often held constant to reduce condensation but Often held constant to reduce condensation but bulb crops prefer alternating temperaturesbulb crops prefer alternating temperatures

• Cultures can be refrigerated to slow growth and Cultures can be refrigerated to slow growth and reduce subculture frequencyreduce subculture frequency

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

• LightLight– IrradianceIrradiance 40 - 80 umol•m 40 - 80 umol•m-2-2•sec•sec-1-1 at culture at culture

level (in a greenhouse the irradiance levels level (in a greenhouse the irradiance levels range from 600 - 1200 umol•mrange from 600 - 1200 umol•m-2-2•sec•sec-1-1 ) )

– RememberRemember: cultures are heterotrophic, : cultures are heterotrophic, therefore high light for photosynthesis is not therefore high light for photosynthesis is not critical. High sucrose levels and low COcritical. High sucrose levels and low CO22

levels inhibit photosynthesislevels inhibit photosynthesis

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation– PhotoperiodPhotoperiod: typically 12 - 16 hours: typically 12 - 16 hours

– Light qualityLight quality: typically cool-white : typically cool-white fluorescent lamps usedfluorescent lamps used

• Vessel and lid effects light quality reaching the Vessel and lid effects light quality reaching the cultureculture

• Incandescent (red) light increases shoot Incandescent (red) light increases shoot elongationelongation

• Fluorescent (blue) light reduces shoot elongationFluorescent (blue) light reduces shoot elongation

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation

• Gases:Gases:– OO22, CO, CO22 and C and C22HH22 all affect the culture all affect the culture

• Problems in tissue cultureProblems in tissue culture– Hyperhydricity (vitrification)Hyperhydricity (vitrification)

• Water-soaked appearance from excess cell waterWater-soaked appearance from excess cell water• Leads to culture deteriorationLeads to culture deterioration• Remedy: change agar type and concentration, Remedy: change agar type and concentration,

reduce condensation/free waterreduce condensation/free water

Hepa filters over vents on lidsHepa filters over vents on lidsreduce condensation and improvereduce condensation and improvegas exchangegas exchange

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation– Internal pathogens- especially bacteria (can Internal pathogens- especially bacteria (can

culture on a medium containing an culture on a medium containing an antibacterial agent)antibacterial agent)

– Release of phenolics (causes blackening of Release of phenolics (causes blackening of the medium). Can be controlled by adding the medium). Can be controlled by adding activated charcoal to the mediumactivated charcoal to the medium

– Tissue proliferation (TP)Tissue proliferation (TP)• Gall-like growths on micropropagated plants Gall-like growths on micropropagated plants

(especially rhododendrons)(especially rhododendrons)

Systems used to regenerate Systems used to regenerate plantlets by micropropagationplantlets by micropropagation– HabituationHabituation

• Cultures (shoots) continue to proliferate even Cultures (shoots) continue to proliferate even when moved to a medium without growth when moved to a medium without growth regulatorsregulators

– Variation in micropropagated plantsVariation in micropropagated plants• Increased vigor - not known whyIncreased vigor - not known why

• Increased branching - in herbaceous plants Increased branching - in herbaceous plants especiallyespecially

• Genetic variation - especially of chimeric plants Genetic variation - especially of chimeric plants like Hostalike Hosta

Stabilization of culturesStabilization of cultures

Determining the proper amounts of cytokininsDetermining the proper amounts of cytokinins

Determining the proper amounts of cytokininsDetermining the proper amounts of cytokinins

Peony embryo excision and placement in tissue culturePeony embryo excision and placement in tissue culture

Chionanthus virginicusChionanthus virginicus embryo excision embryo excision and placement in tissue cultureand placement in tissue culture

Growth after 2 yearsfrom seed

After 2 years from TC

MicrograftingMicrografting

Lack of epicuticular waxesLack of epicuticular waxes

Phenolic build-up in mediumPhenolic build-up in medium

Problems in tissue cultureProblems in tissue culture

Problems in tissue cultureProblems in tissue culture

Difficulties in shoot production in Difficulties in shoot production in Gymnocladus dioicusGymnocladus dioicus““kentuky coffeetree”kentuky coffeetree”

Sources for supplies/info.Sources for supplies/info.

http://aggie-horticulture.tamu.edu/tisscult/microprop/http://aggie-horticulture.tamu.edu/tisscult/microprop/microprop.htmlmicroprop.html

Storage of culture in refrigerationStorage of culture in refrigeration