The role of pregrowth and preculture treatments in terms of both medium composition and exposure duration on survival of embryonic axes of Citrus madurensis after cryopreservation using the vitrification procedure was investigated. The optimal pregrowth treatment for excised embryonic axes was a 3-day treatment with 0.1M sucrose. Preculture was also essential in increasing survival after cryopreservation. Among the various media and treatment durations evaluated, a 24h-preculture of embryonic axes on medium with 0.3M sucrose and 0.5M glycerol was found to be optimal. Using these pregrowth and preculture conditions followed by treatment at 25 degrees C for 20 min each with a loading solution (0.4M sucrose + 2.0M glycerol) and then the PVS2 vitrification solution, direct immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2M sucrose solution for 20 min and transfer of embryonic axes on recovery medium, 82.5% survival and regrowth without intermediary callus formation were obtained with C. madurensis embryonic axes.
The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of intermediate oily seeds using Citrus as a model.
This paper investigates the importance of loading and treatment with a vitrification solution on the survival of Citrus madurensis embryonic axes cryopreserved using a vitrification protocol. Among the seven different loading solutions tested, the solution containing 2 M glycerol + 0.4 M sucrose was the most efficient. Of the six vitrification solutions tested, the PVS2 vitrification solution, applied for 20 min at 25 degree C or for 60 min at 0 degree C, ensured the highest survival. A three-step vitrification protocol, involving the treatment of embryonic axes at 0 degree C with half strength PVS2 solution for 20 min then with full strength PVS2 for an additional 40 min was more efficient than a two-step protocol that involved treatment of axes directly with full strength PVS2 solution for 60 min. After rapid immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2 M sucrose solution for 20 min, culture on solid medium with 0.3 M sucrose for 1 day and growth recovery for 4 weeks on standard medium, survival of C. madurensis embryonic axes reached 85 % following the three-step process, compared with 70 % for the two-step process.
The desiccation and freezing tolerance of seeds, with and without testas, and embryonic axes of Citrus aurantifolia were investigated. Seeds were desiccated with silica gel, under the laminar air flow cabinet or by placing them on a laboratory bench. Whatever the desiccation method employed, survival before and after cryopreservation was higher for seeds without testas. When freezing intact seeds, the highest survival percentage (41.3 %) was achieved after desiccation to 7.3 % moisture content (fresh weight basis) on the laboratory bench. Survival of seeds cryopreserved without testas could reach up to 85 % after desiccation under the laminar air flow cabinet or on the laboratory bench, corresponding to moisture contents of 7.1 and 4.5 %, respectively. After desiccation with silica gel, survival reached a maximum of 60.0 %, for a seed moisture content of 3.3 %. Survival of control embryonic axes was high (80-100 %) whatever the sucrose concentration in the preculture medium and the duration of the desiccation period. After cryopreservation, no survival was noted with embryonic axes, which had not been precultured nor desiccated. Survival of non-desiccated embryonic axes after cryopreservation increased progressively in line with increasing sucrose concentrations in the preculture medium, from 7.5 % with 0.1 M sucrose to 77.5 % with 0.7 M sucrose. Survival of desiccated and cryopreserved embryos was always high, whatever the preculture treatment and desiccation period, ranging from 55.8 % to 92.5 %.
In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation-dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content.