ORIGINAL PAPER Cryopreservation of embryogenic cell suspensions of Catharanthus roseus L. (G) Don. Samar Fatima Æ A. Mujib Æ S.A. Nasim Æ Z.H. Siddiqui Received: 21 October 2008 / Accepted: 31 March 2009 / Published online: 18 April 2009 Ó Springer Science+Business Media B.V. 2009 Abstract An efficient cryopreservation protocol was established for embryogenic cell suspension cultures of Catharanthus roseus. This involved a vitrification-based cryopreservation method wherein embryogenic cells were exposed to a preculture/pretreatment medium prior to their immersion in liquid nitrogen. These cell suspension cul- tures were first initiated from friable embryogenic callus derived from hypocotyls of C. roseus on a medium con- taining 4.52 lM 2,4-Dichlorophenoxy acetic acid (2, 4-D). Among different sucrose (0.09–0.6 M) and sorbitol (0.2– 0.6 M) levels evaluated during preculture, 0.4 M sucrose promoted highest cellular regrowth. Whereas, among pre- treatments Dimethyl sulphoxide (DMSO) (5 or 10%) and glycerol (5 or 10%) at six different levels either alone or in combinations (PT 1–PT 6), the cryopreservation treatment combinations of either 0.4 M sucrose, 5% DMSO, and 5% glycerol (PT-5) or 0.4 M sucrose and 5% DMSO (PT-1) resulted in the highest frequency of viability of embryo- genic cultures. However, the PT-1 treatment produced highest number of cell colonies (10.06 ± 0.55) following reculture of cryopreseved cultures. All calluses regrown in an optimized medium, containing 6.62 lM 6-benzylade- nine (BA) and 5.37 lM a-naphthaleneacetic acid (NAA), resumed normal growth, and produced somatic embryos similar to those from non-frozen embryogenic cultures. These somatic embryos were converted into regenerated plantlets, and all plantlets exhibited normal morphology. Keywords Cryopreservation Á Embryogenic suspensions Á Vitrification solution Á Pre culture Á Pre treatment Abbreviations BA 6-Benzyladenine DMSO Dimethylsulfoxide MS Murashige and Skoog medium NAA a-Naphthaleneacetic acid LN Liquid nitrogen 2,4-D 2,4-Dichlorophenoxy acetic acid Introduction Catharanthus roseus L. (G.) Don. is an important medici- nal plant producing two important anticancer alkaloids, vincristine and vinblastine. However, the natural yield of these alkaloid compounds is quite low, thus several approaches, including biotechnological methods, have been pursued to enhance yield (Moreno et al. 1995; Van der Heijden et al. 2004). Induction of somatic embryogenesis (SE) has been recently reported in C. roseus (Junaid et al. 2006), and germinated somatic embryos as well as leaves derived from these plantlets exhibited higher vincristine contents than those from corresponding in vivo tissues (Junaid et al. 2008). Therefore, maintaining and preserving selected and superior lines of SE cultures, overproducing some of the high-value alkaloid compounds, for long-term durations would be highly desirable. Cryopreservation, storage of living tissues at ultra-low temperatures (-196°C) in liquid nitrogen, has long been used to preserve plant germplasm for either short- or long- term durations without loss of genetic potential (Benson and S. Fatima Á A. Mujib (&) Á S.A.Nasim Á Z.H.Siddiqui Cellular Differentiation and Molecular Genetics Section, Department of Botany, Hamdard University, New Delhi 110062, India e-mail: amujib3@yahoo.co.in 123 Plant Cell Tiss Organ Cult (2009) 98:1–9 DOI 10.1007/s11240-009-9532-z