Cryopreservation of Transformed (Hairy) Roots ofArtemisia annua

Abstract

The antimalarial drug artemisinin has been found in transformed (hairy) roots ofArtemisia annua.A protocol was developed to preserveA. annuahairy 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°C for 1 h followed by cooling at 0.09°C/min to 4°C then cooling to −35°C at 0.72°C/min. Vials containing the root tips were then plunged into liquid nitrogen. After thawing in a water bath to 37°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.