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 40°C. The survival rates were 90% for Maesa and 53% for Medicago, which are sufficient to allow implementation in large storage experimental set-ups.
KeywordsMaesa lanceolata Medicago truncatula Hairy roots Cryopreservation Vitrification Encapsulation-dehydration
- Cha-Um S, Kirdmanee C, Huyen PX, Vathany T (2007) Disease-free production and minimal-growth preservation of in vitro banana (Musa spp.). In: Hummer KE (ed) Proceedings of the second international symposium on plant genetic resources of horticultural crops, Vols. 1 and 2. Acta horticulurae, Seoul, pp 233–240Google Scholar
- Grout BWW (1995) Introduction to the in vitro preservation of plant cells, tissues and organs. In: Grout BWW (ed) Genetic preservation of plant cells in vitro. Springer, Berlin, pp 1–20Google Scholar
- Maes L, Vanden Berghe D, Germonprez N, Quirijnen L, Cos P, De Kimpe N, Van Puyvelde L (2004) In vitro and in vivo activities of a triterpenoid saponin extract (PX-6518) from the plant Maesa balansae against visceral leishmania species. Antimicrob Agents Chemother 48:130–136. doi:10.1128/AAC.48.1.130-136.2004 PubMedCrossRefGoogle Scholar
- Sakai A (2000) Development of cryopreservation techniques. In: FaT Engelmann H (ed), Cryopreservation of tropical germplasm. Current research progress and application. JIRCAS, Rome, pp 1–7Google Scholar
- Yoshimatsu K (2000) Cryopreservation of medicinal plant resources: retention of biosynthetic capabilities in transformed cultures. In: FaT Engelmann H (ed) Cryopreservation of tropical germplasm. Current research progress and application. JIRCAS, Rome, pp 77–90Google Scholar