Preparative Procedures and Culture Medium Affect the Success of Cryostorage of Holostemma annulare Shoot Tips

Shoot tip cryopreservation of Holostemma annulare, an endangered medicinal plant was carried out using Murashige-Skoog (MS) medium containing mM NH⁺ ₄+NO^– ₃; 20.6+39.4 (MS-1), 2.6+18.8 (MS-2) or 0.0+18.8 (MS-3). The three media combinations were tested during four preparative procedures viz.: development of cultures; preconditioning of shoot tip cuttings; preculture of encapsulated shoot tips; and post-freeze recovery to understand the most critical phase of NH₄NO₃ sensitivity. MS-1 used during the initial three preparative steps supported 10.9–16.6% post-freeze recovery of cryopreserved shoot tips. Development of culture in MS-1 and subsequent passages (2nd, 3rd and 4th preparative steps) in MS-2 or MS-3 improved the recovery rate to 26.4–35.8%. MS-3 used throughout the steps favoured 38.5% recovery. Shoot tips from shoot cultures raised in MS-2 upon preconditioning in MS-2 or MS-3 and subsequent preculture of encapsulated shoot tips and post-freeze recovery culture in MS-3 showed maximum regeneration (55%). MS-2 used throughout the procedure supported 48% regeneration of cryopreserved shoot tips.