Cryopreservation of in vitro-grown shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree

Cryopreservation of in vitro axillary shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree, was investigated. Axillary buds (c. 1mm in length) excised from 4-week-old in vitro cultures, were pre-cultured on liquid MS medium supplemented with 0.4 M sucrose for 16 h. These were incubated in 2 M glycerol + 0.4 M sucrose for 20 min at 25°C before being dehydrated with PVS2 solution for 40 min The dehydrated shoot tips were directly immersed in LN. Following cryopreservation and after rapid warming at 40°C, shoot tips were quickly washed with MS + 1.2 M sucrose solution for 20 min and then plated on top of filter paper placed on MS medium supplemented with 0.1 mg l⁻¹ BAP, kept in darkness for one day followed by placement of shoots directly on the medium and incubation in darkness for a day more, before transfer of cultures to light. Average survival in terms of normal shoot formation after 4 weeks of plating was 56.6%. The rescued shoot tips were bulked up by subsequent nodal cultures and when put onto 0.02 mg l⁻¹ NAA showed a rhizogenic response. Thus, in vitro-grown shoot tips of Crateva nurvala were successfully cryopreserved following the optimization of the PVS2-vitrification protocol.