In Vitro Conservation Methods in Rain Lilies

Zephyranthes is valued as conventional and native landscaping ornamental plant as well as a traditional Chinese medicinal herb and has been attributed with various pharmacological activities such antidiabetic and anti-HIV. This study was carried out to evaluate the potential of in vitro  conservation methods using the artificial seed and encapsulation-dehydration cryopreservation technique for seed embryos in Zephyranthes atamasca and Z. grandiflora. Seed embryos were selected for encapsulation with different concentration of sodium alginate (3%, 4%, and 5%) and calcium chloride (either 25, 50, 75, and 100 mM) followed by no encapsulated embryo as a control. The greatest viability of encapsulated seeds achieved was 95% in Z. grandiflora and 85% in Z. atamasca with the combination of 4% sodium alginate with 100 mM calcium chloride after 2 weeks at 5°C. After optimizing the gelling agents, embryos were immersed in ½-strength liquid MS medium supplemented with 0, 20, 30, 40, and 50 g/L sucrose for 3 days. The highest viability with A_530nm0.12 and A_530nm0.16 were achieved when embryos were cultured in pretreatment medium with 30 g/L sucrose in Z. grandiflora and Z. atamasca, respectively. Once the optimum concentration of sucrose was determined, the pretreatment duration was evaluated; embryos were pretreated at different intervals (0, 1, 2, 3, 4, and 5 days) with the aim of conditioning them to withstand freezing stress. The greatest viability was observed after 2 days of pretreatment with A_530nm0.76 and A_530nm0.61 in Z. grandiflora and Z. atamasca, respectively. Dehydration under the air current of a laminar air flow cabinet was used after 1, 2, 3, 4, 5, and 6 h. The highest viability by TTC assay after cryopreservation was observed with 54% viability for Z. grandiflora and 48% viability with Z. atamasca, after 2 h of dehydration, whereas the control treatment (0 h drying) showed 9% viability for Z. grandiflora and 7% viability with Z. atamasca viability followed by ~96% viability in non-freezing treatment. Rain lilies embryos were successfully preserved and functioned as an artificial seed in Z. atamasca and Z. grandiflora. In addition, the cryopreservation technique using encapsulation-dehydration method has been established for the rain lilies embryo and can be used for other flowers embryos with few modifications.