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. an nua 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 cryopr otection. They were then treated with a cryoprotecting mixture contain ing 8% (v/v) dimethyl sulfoxide (Me(2)SO) and 20% (w/v) sucrose at 25 degrees C for 1 h followed by cooling at 0.O9 degrees C/min to 4 degre es C then cooling to -35 degrees C at 0.72 degrees C/min. Vials contai ning the root tips were then plunged into liquid nitrogen. After thawi ng in a water bath to 37 degrees C, root tips were held in the cryopro tecting mixture for 10 min before it was diluted to 25% of its origina l 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%. Ind ependent variables in this study included 1) Me(2)SO concentration; 2) the type and concentration of cosolutes; 3) cooling rate(s); 4) the t emperature at which the sample is transferred to liquid nitrogen; 5) t he 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. (C) 1996 Academic Press, Inc.