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Techniques of Micropropagation Chapter 18. Systems used to regenerate plantlets by micropropagation I.) Axillary shoot formationI.) Axillary shoot formation.

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Presentation on theme: "Techniques of Micropropagation Chapter 18. Systems used to regenerate plantlets by micropropagation I.) Axillary shoot formationI.) Axillary shoot formation."— Presentation transcript:

1 Techniques of Micropropagation Chapter 18

2 Systems used to regenerate plantlets by micropropagation I.) Axillary shoot formationI.) Axillary shoot formation –Meristem tip culture Results in plantlets free from viruses, fungi and bacteria (esp. when coupled with heat treatment)Results in plantlets free from viruses, fungi and bacteria (esp. when coupled with heat treatment) Important for many herbaceous crops (carnations, mums, orchids, geraniums, banana, potato, sweetpotato)Important for many herbaceous crops (carnations, mums, orchids, geraniums, banana, potato, sweetpotato) With woody plants, meristems are often graftedWith woody plants, meristems are often grafted –Axillary shoot culture Reliably reproduces the genotype of the parent plant (expands existing buds)Reliably reproduces the genotype of the parent plant (expands existing buds)

3 Carnation meristem

4 Nodal shoot production at cotyledonary stage

5 Systems used to regenerate plantlets by micropropagation Adventitious shoot formationAdventitious shoot formation –Initiated directly on the explant or indirectly from callus –Results in high rates of multiplication –Results in increased aberrant (off-type) plants –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.)

6 Bulblet formation in tissue culture

7 Hosta culture

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10 Types of micropropagation

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12 Systems used to regenerate plantlets by micropropagation III.) Callus, cell & protoplast culture systemsIII.) Callus, cell & protoplast culture systems –Can be subcultured and maintained indefinitely –Callus 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). Parenchyma produces meristems (= meristmoids)Can be induced to form organs (Organogenesis). Parenchyma produces meristems (= meristmoids) First done with tobacco & carrotFirst done with tobacco & carrot

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16 Direct shoot production (organogenesis)

17 Systems used to regenerate plantlets by micropropagation –Cell suspensions Produced from friable callus (= loose)Produced from friable callus (= loose) Maintained in shaker cultures or bioreactorsMaintained in shaker cultures or bioreactors –Protoplast culture Cell culture without cell walls (cellulase added to degrades the cell wall)Cell culture without cell walls (cellulase added to degrades the cell wall) Only plasmamembrane remainsOnly plasmamembrane remains Osmotic pressure must be maintained to keep cells from rupturing (mannitol used)Osmotic pressure must be maintained to keep cells from rupturing (mannitol used) Why done? Secondary plant products that leak from the protoplasts are collected (ex: taxol, sanguinaria)Why done? Secondary plant products that leak from the protoplasts are collected (ex: taxol, sanguinaria)

18 Cell cultures on a shaker

19 Bioreactors for cells or protoplasts

20 Protoplast culture

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24 Sanguinaria canadensis bloodroot

25 Systems used to regenerate plantlets by micropropagation IV.) Somatic embryogenesis & Synthetic seedIV.) Somatic embryogenesis & Synthetic seed –Development of embryos without a zygote (i.e. from non-gamete cells) –Roots and shoots develop simultaneously to form embryoids (i.e.: carrots)

26 Systems used to regenerate plantlets by micropropagation –Arise from: Adventitious somatic embryogenesis (directly from cells = embryogenic cells). Usually arise near zygotic cellsAdventitious somatic embryogenesis (directly from cells = embryogenic cells). Usually arise near zygotic cells Induced somatic embryogenesis. Callus must form first (often in suspension culture). Usually conditioned on high levels of auxin (2,4-D)Induced somatic embryogenesis. Callus must form first (often in suspension culture). Usually conditioned on high levels of auxin (2,4-D) –Uses: Clonal propagationClonal propagation Genetic manipulation -using Agrobacterium tumefasciens or a gene-gunGenetic manipulation -using Agrobacterium tumefasciens or a gene-gun

27 Somatic embryogenesis (soybean)

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29 Somatic embryogenesis (sitka spruce)

30 Systems used to regenerate plantlets by micropropagation Environmental conditions during tissue cultureEnvironmental conditions during tissue culture –Temperature °F °F Often held constant to reduce condensation but bulb crops prefer alternating temperaturesOften held constant to reduce condensation but bulb crops prefer alternating temperatures Cultures can be refrigerated to slow growth and reduce subculture frequencyCultures can be refrigerated to slow growth and reduce subculture frequency

31 Systems used to regenerate plantlets by micropropagation LightLight –Irradiance umolm -2 sec -1 at culture level (in a greenhouse the irradiance levels range from umolm -2 sec -1 ) –Remember: cultures are heterotrophic, therefore high light for photosynthesis is not critical. High sucrose levels and low CO 2 levels inhibit photosynthesis

32 Systems used to regenerate plantlets by micropropagation –Photoperiod: typically hours –Light quality: typically cool-white fluorescent lamps used Vessel and lid effects light quality reaching the cultureVessel and lid effects light quality reaching the culture Incandescent (red) light increases shoot elongationIncandescent (red) light increases shoot elongation Fluorescent (blue) light reduces shoot elongationFluorescent (blue) light reduces shoot elongation

33 Systems used to regenerate plantlets by micropropagation Gases:Gases: –O 2, CO 2 and C 2 H 2 all affect the culture Problems in tissue cultureProblems in tissue culture –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, reduce condensation/free waterRemedy: change agar type and concentration, reduce condensation/free water

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36 Hepa filters over vents on lids reduce condensation and improve gas exchange

37 Systems used to regenerate plantlets by micropropagation –Internal pathogens- especially bacteria (can culture on a medium containing an antibacterial agent) –Release of phenolics (causes blackening of the medium). Can be controlled by adding activated charcoal to the medium –Tissue proliferation (TP) Gall-like growths on micropropagated plants (especially rhododendrons)Gall-like growths on micropropagated plants (especially rhododendrons)

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45 Systems used to regenerate plantlets by micropropagation –Habituation Cultures (shoots) continue to proliferate even when moved to a medium without growth regulatorsCultures (shoots) continue to proliferate even when moved to a medium without growth regulators –Variation in micropropagated plants Increased vigor - not known whyIncreased vigor - not known why Increased branching - in herbaceous plants especiallyIncreased branching - in herbaceous plants especially Genetic variation - especially of chimeric plants like HostaGenetic variation - especially of chimeric plants like Hosta

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47 Stabilization of cultures

48 Determining the proper amounts of cytokinins

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50 Peony embryo excision and placement in tissue culture

51 Chionanthus virginicus embryo excision and placement in tissue culture and placement in tissue culture Growth after 2 years from seed After 2 years from TC

52 Micrografting

53 Lack of epicuticular waxes Phenolic build-up in medium Problems in tissue culture

54 Difficulties in shoot production in Gymnocladus dioicus kentuky coffeetree

55 Sources for supplies/info. Storage of culture in refrigeration


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