Multiplication and cryopreservation of adventitious roots of Cleome rosea Vahl

Abstract

Adventitious root cultures of Cleome rosea were established using root segments from proximal and middle root regions of in vitro-propagated plants. The root explants were cultured in liquid MS medium supplemented with the following growth regulators: naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), and N6-furfuryladenine (KIN). The highest biomass accumulation was obtained with roots, cultured in media with NAA. The use of 2,4-D resulted in callus formation, while explants cultured in the presence of KIN developed buds. Protocols for long-term maintenance of in vitro roots through cryopreservation by vitrification were also evaluated using plant vitrification solution 2 (PVS2). The use of NAA during pre- and post-culture steps was essential to recovery after exposure to liquid nitrogen. On the other hand, the presence or absence of light during culture maintenance had little effect on the process. The highest recovery frequency after cooling in liquid nitrogen (63.6 ± 14.4%) was reached after exposure to PVS2 for 30 min. The cryopreserved roots showed excellent ability to multiply, which remained stable for up to five subcultures. Moreover, these roots also displayed the capacity for shoot induction when cultivated on solid MS medium supplemented with 0.50 mg/L 6-benzyladenine (BA), reaching regeneration frequencies of 60–82% over five subcultures.