Freezing Resistance and Cryopreservation of Cell Strains of Rhaponticum carthamoides and Thalictrum minus

Effects of long-term (few months) culturing and short pregrowth (up to 7 days prior to deep freezing) in the presence of mannitol (5–6%), ABA (5.0–7.5 × 10^–5 M), or both substances on cryogenic resistance of leusea (Rhaponticum carthamoides, strains Rhs-2 and Rhs-8) and meadow rue (Thalictrum minus L., strain B-233) cell suspension cultures were studied. Cryoprotective capacities of 48 solutions were studied at slow (0.33°C/min) freezing to the temperature of liquid nitrogen with an automatic initiation of crystallization. Cells were stored in liquid nitrogen for several days or months. ABA had a cryoprotective effect, provided that subculturing intervals were 12–14 days. At more frequent subculturing (every 7 days), pregrowth on ABA-containing medium did not increase survival percentage compared to pregrowth with mannitol. Successful cryopreservation of these strains has been achieved due to strict standardization of 7-day subculturing regime, pregrowth in the presence of mannitol prior to freezing, and cryopreservation with dimethyl sulfoxide, sucrose, trehalose, and glycerol. The cell survival rates after thawing were 60% (Rhs-8), 80% (Rhs-2), and 70% (B-233). The cell growth resumed on the third to seventh day. The growth indices and protoberberine synthesizing activity in B-233 strain reached their control values at the ninth subculturing after a post-thaw recovery.