Short-term Storage of Seeds and Cryopreservation of Embryonic Axes of Lepisanthes fruticosa

Abstract

Aims: This work highlights short-term storage of recalcitrant Lepisanthes fruticosa seeds and long-term conservation attempts of its embryonic axes (EAs) through cryopreservation. Study design: This study adopted the Completely Randomized Design (CRD). Ten samples were used for each experiment and replicated for 3 – 5 times. Place and Duration of Study: Cryopreservation Laboratory, Agrobiodiversity and Environment Research Centre, MARDI Headquarters, Malaysia, in 2017 and 2018. Methodology: Short-term storage was carried out using fresh seeds at 54% moisture content and stored at 8±1 °C and 25±2 °C for 7 weeks. Three variations to sterilization were attempted to optimize survival while keeping contamination low. Cryopreservation using two different methods were tested, namely vitrification and the encapsulation vitrification method. Vitrification technique involved the pre-culturing of EAs overnight in different sucrose pre-culture concentrations (0, 0.2, 0.4 and 0.6 M) prior to, loading, dehydration with vitrification solution (PVS2), rapid immersion into liquid nitrogen (-196°C), rapid warming, unloading and recovery. While, encapsulation vitrification involved encapsulation of the EAs using 3% sodium alginate followed by exposure to different duration (0, 10, 20, 30, 40 and 50 minutes) of vitrification solution (PVS2) prior to cryopreservation. Results: L. fruticosa seeds can be safely stored for short-term up to 7 weeks of storage either at 8±1 °C or 25±2 °C with no loss in germination. This study also showed that EA was amenable to cryopreservation and 13.33 – 66.67% of viability was obtained when the EAs were cryopreserved using the vitrification technique. The best result was obtained with 66.67% viability, when the EAs were pre-cultured with 0.4M sucrose prior to exposure to PVS2 and liquid nitrogen. Cryopreservation of EAs using the encapsulation-vitrification method was unsuccessful. Conclusion: Seeds of L. fruticosa can be stored for short-term (up to 7 weeks) using hydrated/non-dried seeds where they can be successfully stored at 8±1 °C and 25±2 °C for up to 7 weeks. For long-term (cryopreservation), EAs can be cryopreserved upon pre-culture with 0.4M sucrose prior to exposure to PVS2 and liquid nitrogen through vitrification technique.