From leaf explants to hanging rooted plantlets in a mist reactor

Abstract

A mist bioreactor using a disposable bag as a culture chamber was used to propagate single nodal explants of Artemisia annua into rooted plantlets that were ready for soil using a single batch (one-step) culture. To vertically scale plant growth inside the mist reactor, poly-l-lysine (PLL)-coated 70 µm nylon mesh and solid polypropylene sheeting were used for explant attachment. Both manually chopped and blender-chopped (blenderized) shoot tissues were attached to PLL-coated substrates. Compared to blenderized shoots, manually chopped tissues were larger with better attachment to PLL-coated substrates. Regardless of substrates or explant preparation method, 80–95 % of initially attached shoot tissues remained attached to PLL-coated surfaces after being misted with culture medium for 24 h. New shoot proliferation increased about tenfold as the size of blenderized shoot tissue increased. To reduce callusing during shoot proliferation and thus stimulate root initiation, original shooting medium was reduced to half strength of phytohormone and Murashige and Skoog salts. The duration of shoot proliferation was also reduced from 2 to 1 week. Original rooting medium was then further improved with NAA or IBA. After successive shooting, rooting and in vitro acclimatization, the nodal explants attached to PLL-coated hanging strips and developed into fully rooted plantlets in the mist reactor. Although most of the large rooted plantlets detached from the hanging strips by the time of harvest, they had fully functional stomata and were later successfully established in the soil, suggesting this “hanging garden” technology may prove useful for micropropagation.