Excised embryonic axes from seeds of three taxa, namely, Citrus suhuiensis cv. limau madu, Citrumelo (Citrus paradisi x Poncirus trifoliate) and Fortunella polyandra, were desiccated in a laminar airflow, over silica gel, and ultra-rapidly. Desiccation sensitivity (WC50) was estimated for each taxon using the quantal response model. High desiccation tolerance (WC50 = 0.11 g water per g dry mass. g/gdw) was observed for limau madu embryonic axes desiccated in a laminar airflow and ultra-rapidly (WC50 =0.10 g/gdw). Desiccation tolerance was substantially lower (WC50 = 0.19 g/gdw) for silica gel dehydration. Similarly, high desiccation tolerance (WC50 = 0.15 g/gdw) was associated with F. polyandra embryonic axes when desiccated in a laminar airflow, while a lower desiccation tolerance (WC50 = 0.17 g/gdw) was observed with silica gel dehydration. Ultra-rapid desiccation led to the highest desiccation tolerance (WC50 = 0.14 g/gdw). The dehydration rate, however, had no influence on desiccation tolerance (WC50 ~ 0.14 g/gdw) for Citrumelo embryonic axes. After each desiccation period, embryonic axes were directly immersed in liquid nitrogen (LN) followed by rapid rewarming. Normal seedling recovery of 80 to 83% for excised embryonic axes of limau madu was observed for laminar airflow and ultra-rapid dehydration, but for silica gel dehydration, 57% recovery was obtained. Similarly, for Citrumelo, high recoveries of 100% and 97% were obtained from axes desiccated in a laminar airflow and using ultra-rapid dehydration, respectively, whereas a lower value was associated with silica gel dehydration (80%). For F. polyandra, 50% recovery was obtained both for laminar airflow and ultra-rapid dehydration, while much lower recovery (43%) was associated with silica gel dehydration. Regardless of the drying method employed, axis survival percentages following exposure to LN were commensurate with the desiccation sensitivity pattern.
Following the investigation of desiccation sensitivity and freezing tolerance of the whole seed of Citrus suhuiensis cv. limau langkat, desiccation sensitivity and cryopreservation of the excised embryonic axes from the seeds of the same species were examined. Three drying conditions were employed: desiccation by equilibrium for the whole seeds and desiccation in laminar airflow and over silica gel for the excised embryonic axes. The relevance of desiccation sensitivity (WC50) to cryopreservation of whole seeds and excised axes was investigated. High desiccation tolerance (WC50 = 0.034 g H2O x g(-1)dw) was acquired for axes desiccated with faster dehydration rate (1.5 g x g(-1) x h(-1)) in laminar airflow compared to substantially lower desiccation tolerance (WC50 = 0.132 and 0.110 g H2O x g(-1)dw) acquired under slower dehydration rates (1.0 and 0.005 g x g(-1) x h(-1)) for axes desiccated over silica gel and whole seeds desiccated by equilibrium respectively. While few whole seeds (8.3%) survived freezing, high recovery percentages of axes (83.3% and 62.2%) after freezing were obtained under laminar airflow and silica gel drying conditions respectively. Irrespective of the drying method employed, axes survival percentages after exposure to LN temperature commensurate with the desiccation sensitivity pattern. For the whole seeds, a factor other than desiccation sensitivity that limits the tolerance to exposure to LN temperature seems to exist and still needs to be defined.
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.
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.
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.