Cryopreservation of hairy root cultures of Maesa lanceolata and Medicago truncatula

To study the production of secondary metabolites of Maesa lanceolata and Medicago truncatula, hairy root cultures of both plant species were established. Because maintenance of large numbers of cultures is laborious and costly, we developed a cryopreservation protocol and stored different isolated lines over time. Using encapsulation-dehydration, high survival rates were observed for both Maesa and Medicago hairy roots. Root tips were isolated and encapsulated in calcium-alginate beads, containing 0.1 M sucrose. The encapsulated hairy roots were precultured for 3 days using basal medium containing high sucrose concentrations. Medicago root tip growth during the preculturing time lead to unwanted outgrowth which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated in the air flow of a laminar flow until 35-40% of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and after different storage times thawed in a water bath at 40A degrees C. The survival rates were 90% for Maesa and 53% for Medicago, which are sufficient to allow implementation in large storage experimental set-ups.