Cryopreservation of transformed (hairy) roots of Artemisia annua

The antimalarial drug artemisinin has been found in transformed (hairy) roots of Artemisia annua. A protocol was developed to preserve A. annua hairy roots in liquid nitrogen. Root tips were excised from 7-day-old cultures and held on solid White's medium for 24 h prior to cryoprotection. They were then treated with a cryoprotecting mixture containing 8% (v/v) dimethyl sulfoxide (Me2SO) and 20% (w/v) sucrose at 25 degrees C for 1 h followed by cooling at 0.09 degrees C/min to 4 degrees C then cooling to -35 degrees C at 0.72 degrees C/min. Vials containing the root tips were then plunged into liquid nitrogen. After thawing in a water bath to 37 degrees C, root tips were held in the cryoprotecting mixture for 10 min before it was diluted to 25% of its original concentration. Root tips were washed once with fresh liquid White's medium and held for 1 h prior to culturing on White's medium with 0.2% Gelrite and 3% (w/v) sucrose. Regrowth of root tips averaged 65%. Independent variables in this study included 1) Me2SO concentration; 2) the type and concentration of cosolutes; 3) cooling rate(s); 4) the temperature at which the sample is transferred to liquid nitrogen; 5) the age of the culture from which root tips are taken; 6) the recovery period between root tip excision and immersion in cryoprotectant; and 7) the amount of Gelrite used in the postthaw plating medium.