From cells to embryos to rooted plantlets in a mist bioreactor

A mist bioreactor using a disposable bag as culture chamber was used to propagate carrot embryogenic cells into rooted plantlets. The best operating configuration was akin to a vertical hanging garden using 50–90 μm nylon mesh for explant attachment. Cells spray inoculated into the reactor were 51.2 % viable. Misting cycle and aeration conditions were studied and showed that under the same hourly volumetric nutrient feed and 0 VVM, embryo development in the reactor was best using a 0.3 min on/2.7 min off misting cycle, yielding about 23 % post heart stage embryos. Compared to 0 VVM, 3 % CO2 enrichment improved embryo development in reactor culture. Spray inoculated cells also attached to several vertically hung poly-l-lysine coated strips and then developed in situ into embryos. Cell attachment was significantly improved when they were suspended in salt-free sucrose solution during spray inoculation. Almost 90 % of the originally attached cells remained on the nylon mesh 24 h later after spraying with B5 medium in the mist reactor. Strip grown embryos had the same post heart stage ratio but shorter overall length compared to those developed on a horizontal platform. Young plantlets developed uniformly up and down the hanging strips and did not detach after 3 weeks of culture suggesting this technology may prove useful for improving micropropagation.