Techniques of Micropropagation Chapter 18

Systems used to regenerate plantlets by micropropagation I.) Axillary shoot formation Meristem tip culture 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) With woody plants, meristems are often grafted Axillary shoot culture Reliably reproduces the genotype of the parent plant (expands existing buds)

Carnation meristem

Nodal shoot production at cotyledonary stage

Systems used to regenerate plantlets by micropropagation Adventitious 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) Cotyledons (ie: conifers) Immature inflorescence (ie: Hosta and daylily) Bulb scales (ie: Easter lily, hyacinths, etc.)

Bulblet formation in tissue culture

Hosta culture

Hosta culture

Hosta culture

Types of micropropagation

Systems used to regenerate plantlets by micropropagation III.) Callus, cell & protoplast culture systems Can be subcultured and maintained indefinitely Callus culture Produced in response to wounding & hormones Almost all plant parts can be induced to produce callus Both auxins & cytokinins must be in the medium Can be induced to form organs (Organogenesis). Parenchyma produces meristems (= meristmoids) First done with tobacco & carrot

Direct shoot production (organogenesis)

Systems used to regenerate plantlets by micropropagation Cell suspensions Produced from “friable” callus (= loose) Maintained in shaker cultures or bioreactors Protoplast culture Cell culture without cell walls (cellulase added to degrades the cell wall) Only plasmamembrane remains 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)

Cell cultures on a shaker

Bioreactors for cells or protoplasts

Protoplast culture

Protoplast culture

Sanguinaria canadensis “bloodroot”

Systems used to regenerate plantlets by micropropagation IV.) 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)

Systems used to regenerate plantlets by micropropagation Arise from: Adventitious 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) Uses: Clonal propagation Genetic manipulation -using Agrobacterium tumefasciens or a gene-gun

Somatic embryogenesis (soybean)

Somatic embryogenesis (soybean)

Somatic embryogenesis (sitka spruce)

Systems used to regenerate plantlets by micropropagation Environmental conditions during tissue culture Temperature 68 - 81°F Often held constant to reduce condensation but bulb crops prefer alternating temperatures Cultures can be refrigerated to slow growth and reduce subculture frequency

Systems used to regenerate plantlets by micropropagation Light Irradiance 40 - 80 umol•m-2•sec-1 at culture level (in a greenhouse the irradiance levels range from 600 - 1200 umol•m-2•sec-1 ) Remember: cultures are heterotrophic, therefore high light for photosynthesis is not critical. High sucrose levels and low CO2 levels inhibit photosynthesis

Systems used to regenerate plantlets by micropropagation Photoperiod: typically 12 - 16 hours Light quality: typically cool-white fluorescent lamps used Vessel and lid effects light quality reaching the culture Incandescent (red) light increases shoot elongation Fluorescent (blue) light reduces shoot elongation

Systems used to regenerate plantlets by micropropagation Gases: O2, CO2 and C2H2 all affect the culture Problems in tissue culture Hyperhydricity (vitrification) Water-soaked appearance from excess cell water Leads to culture deterioration Remedy: change agar type and concentration, reduce condensation/free water

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

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)

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

Stabilization of cultures

Determining the proper amounts of cytokinins

Determining the proper amounts of cytokinins

Peony embryo excision and placement in tissue culture

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

Micrografting

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

Difficulties in shoot production in Gymnocladus dioicus Problems in tissue culture Difficulties in shoot production in Gymnocladus dioicus “kentuky coffeetree”

Sources for supplies/info. http://aggie-horticulture.tamu.edu/tisscult/microprop/microprop.html Storage of culture in refrigeration