Cryopreservation Of Sour Orange (Citrus Aurantium) Shoot Tips By Encapsulation-Dehydration

Ex-situ conservation of the Citrus has been considered as problematic and early reports have found that Citrus was a sub orthodox species and cannot be stored at low temperature. Field genebanks are difficult to maintain due to pest, disease and climate hazards. In-vitro culture methods also provide only short-term storage but this is difficult and time-consuming. For a long-term storage of plant germplasm, cryopreservation is currently the best option. Therefore a cryopreservation protocol needed to be developed to conserve Citrus germplasm for long-term. 2, 3, 5-Triphenyl tetrazolium (TTC) test was used for an assessment of cell survival after cryopreservation at 490nm. The shoot tips were encapsulated and osmoprotectant on a shaker at 100 rpm with two sucrose concentrations (0.5M & 0.75M). Subsequently, encapsulated beads were dehydrated under laminar air flow and silica gel for 6 hours. The encapsulated beads were plunged directly into liquid nitrogen for a minimum of 48 hours. Encapsulated beads were thawed at 400C for 2 minutes and rehydrated using liquid MS for 10 minutes. The beads were transferred to re-culture media optimized in experiment 2. Then cultures were kept in dark for 2 days and 1 day in semi light condition to avoid photo-oxidative stress. High viability could be seen when used mature shoots & encapsulated using 4% sodium alginate. The best condition for the encapsulation-dehydration of Citrus aurantium was obtained when beads were pretreated with the osmoprotection medium for 20 hours and dehydrated for 6 hours. Beads were pretreated with 0.75M sucrose and dehydrated under laminar air flow recorded the maximum survival (21.6%). MS medium supplemented with 2mg/l BAP was used as a re-culture medium with 56.5% survival. Finally, beads were pretreated with 0.75M sucrose and dehydrated under laminar air flow method was appropriate to cryopreservation of Citrus aurantium within the tested range.

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