A variety of different bioreactors have been developed for use in initiating and cultivating somatic embryos. The various designs for embryogenesis and culture are critically evaluated here. Bioreactor optimization and operation methods are also described along with recommendations for use based on desired outcome.

Bioreactors for Plant Embryogenesis and Beyond

The medicinally important plant Artemisia annua was used as an example to study the effect of mist reactor conditions on shooting, rooting, and in vitro acclimatization. During all three stages, plants grown in the mist reactor yielded more shoot biomass than corresponding gelled medium controls. During shoot development, shooting percentage and shoot multiplication increased with increased light intensity and CO2. Shooting percentage and shoot multiplication were also better under a once vs. twice per hour misting frequency. Hyperhydricity was lowest under light and CO2 enrichment and with less frequent misting. Compared to unhyperhydric shoots, hyperhydric shoots showed significantly greater water content. Hyperhydric shoots also produced fewer glandular trichomes and artemisinin and flavonoids were 3 and 14.5%, respectively, compared to unhyperhydric shoots. During root development, all aspects of root growth, including rooting percentage, number of primary roots and secondary roots, and length of the latter, improved with more frequent misting cycles and light and CO2 enrichment. Although plants grown in the mist reactor did not yield a better rooting percentage than gelled medium controls, new roots formed in the reactor were thicker than those grown on gelled medium. During in vitro acclimatization, reducing the relative humidity of the ventilating air from ~50 to ~10% improved stomatal closure and subsequent ex vitro survival. Plantlets acclimatized in unventilated reactors had broken stems with less lignin. Together, these results showed that compared to traditional methods using gelled medium, the mist reactor can be used to improve micropropagation.

From leaf explants to rooted plantlets in a mist reactor

A mist reactor was used to study plant growth and development under various environmental conditions towards the production of healthy plantlets ready for soil transplant in one step from inoculation. In addition, a 3D type of cultivation via surface attachment of explants to vertically hanging strips inside the mist reactor was also investigated to maximize productivity with minimal footprint. Using carrot as the model species, pre-embryogenic cell suspensions were successfully spray-inoculated onto hanging poly-L-lysine (PLL)-coated nylon mesh to which they then attached and remained for several weeks while they developed into rooted plantlets. To study single step micropropagation from shoot explants to fully acclimatized plantlets, Artemisia annua was used as the model species. Nodal cuttings of A. annua were inoculated onto PLL-coated mesh strips by briefly immersing the strips in the suspension of nodal cuttings. Investigation of medium, phytohormones, CO2, ventilation level and humidity ensued resulting in selection of a preferred final process that reduced physiological aberrations like hyperhydricity and was time efficient. The nodal cuttings that attached to the strips were first misted with half strength shooting medium for 7 days to develop new shoots. Then the new shoots were misted with the rooting medium supplemented with NAA for 12 days to develop roots. Rooted plantlets were acclimatized in the same rooting medium for 9 days to acquire fully functional stomata prior to planting into soil. Taken together this study suggested that fully developed plantlets ready for planting into soil could be obtained in a single step in a bioreactor from embryogenic cells or from nodal explants.

Towards automating micropropagation: from cells to shoots to plants in one step

Camellia fascicularis is an endangered evergreen ornamental plant with pale yellow flowers. An efficient and reproducible in vitro regeneration method is required for its large-scale propagation an...

In vitro propagation of Camellia fascicularis: a plant species with extremely small populations

Hyperhydricity or glassiness is considered as a frequent problem associated with the in vitro growth and development of watermelon ( Citrulus lanatus , cv. Giza 1). Explants were cultured on MS (Murashige and Skoog, 1962) medium containing 6-bensyladenine (BA), kinetin (Kin) or thidiazuron (N-phenyl N 1,2,4-chlorophenoxyacetic acid) (TDZ) each applied at 0, 0.25, 0.5, 1, 2 and 4 mg L -1 ). The highest number of regenerating shoots per explants MS medium containing 0.5 mg/l TDZ proved to be the most efficient in the induction of adventitious organogenesis, while BA applied at 2 mg L -1 was the most efficient in axillary shoot proliferation. Increase in BA concentration (above 2 mg L -1 ) gave multiple shoots and led to more pronounced hyperhydricity, while the use of 1 mg L -1 facilitated single-shoot growth with relatively lower incidence of hyperhydricity. Substitution of agar (10 g L -1 ) with gelrite (4 g L -1 ) in the medium as well as improving vessel aeration reduced occurrence of hyperhydricity. Provision for aeration was relatively superior to other approaches, but it led to faster dehydration of medium and limited growth of cultures. Rooted shoots produced on half strength MS medium with aeration, and 0.5 mg L -1 indole 3-butyric acid (IBA) were transferred to pots and acclimatized in green-house with a survival rate of 80%. Keywords: Aeration, gelling agents, hyperhydricity, in vitro , rooting, plant growth regulators, ventilation, watermelon

/pdf/minimizing-the-hyperhydricity-associated-with-in-vitro-11u9pnyh6c.pdf

Minimizing the hyperhydricity associated with in vitro growth and development of watermelon by modifying the culture conditions

Glandular trichomes on leaf epidermis of Artemisia annua was a major site for artemisinin production. The influence of culture vessel fitted with or replaced with perforated cap was investigated on growth and microstructures of in vitro A. annua leaves. Increment in height and fresh biomass of plantlets regardless of clones (TC1, TC2 and Highland) in culture vessel fitted with normal cap were significantly higher than those cultures with perforated cap after eight weeks of culture. Desiccation of the culture medium was observed in culture vessels fitted with or replaced with perforated caps. Three clones showed different growth profile and sensitivity toward ventilation. T-shaped trichome (non-glandular) and capitates trichome (glandular trichome) were found on adaxial and abaxial epidermis of A. annua leaves. Using perforated cap induced more trichomes on the adaxial surface of leaf for all three tested clones. Stomata of plantlets cultured in culture vessels with normal caps were always opened and opposite trend observed in culture vessels replaced with perforated caps. Results indicated that ventilation due to cap type influenced the growth of in vitro plantlets and microstructures of A. annua leaves.

Effect of perforations of culture vessel cap on growth and leaf microstructure of in vitro plantlets of Artemisia annua L.

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Effect of perforations of culture vessel cap on growth and leaf microstructure of in vitro plantlets of Artemisia annua L.