This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of strawberry (Fragaria × ananassa Duch.) cvs. ‘Wonkyo3114’ and ‘Gurumi40’. The shoot tips were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.5M). Precultured explants were osmoprotected with loading solution (LS, C4) containing 20% glycerol and 20% sucrose for 40 min and exposed to dehydration solution (B5) containing 40% glycerol and 40% sucrose for 40 min at 25℃, Subsequently, the explants were transferred onto droplets containing 2.5 μL PVS3 on sterilized aluminum foils (4 ㎝ × 0.5 ㎝) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regrowth rate (%) in both the cultivars was obtained when the shoot tips were precultured with MS + 0.3M sucrose for 40 h at 25℃. The cryopreserved shoots tips exhibited 55% regrowth rate by culturing in NH4NO3-free MS medium supplemented with 3% sucrose, 1.0 g/L casein, 1.0㎎/L GA3, and 0.5 ㎎/L BA for 5 weeks and in MS medium supplemented with 0.5 ㎎/L GA3 for 8 weeks. This result shows that droplet-vitrification could be employed as a promising method for cryostorage of strawberry germplasm.

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: ‘Frost Eureka limon’ and ‘Cook Eureka limon’, using a droplet-vitrification method. The shoot tips that were excised from in vitro grown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at 25℃ and incubated in a loading solution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at 25℃. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at 0℃ or PVS3 at 25℃. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in ½ MS for 30 min at 25℃. Shoot tips were post-cultured overnight on survival medium and then micrografted onto ‘trifoliate orange’ (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% for cryopreserved shoot tips of ‘Frost Eureka limon’ and ‘Cook Eureka limon’, respectively, when pre-cultured in 0.3 M and 0.5 M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at 0℃. We also investigated whether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoot tips. The viability of cooled samples, following culturing on woody plant media (WPM) containing ¼ ammonium nitrate overnight before micrografting, was the highest (70.3%) in ‘Frost Eureka limon’. The study described here is a cost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruit plants and for breeding disease resistance.

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of Chrysanthemum morifolium (Ramat.) cvs. ‘Borami’ and ‘Yes morning’. The shoot tips of Chrysanthemum were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explants were treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed to dehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at 25℃, and then transferred onto droplets containing 2.5 ㎕ PVS3 on sterilized aluminum foils (4 ㎝ × 0.5 ㎝) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at 25℃ in both the cultivars. The viability of cooled samples, followed by culturing on NH4NO3-free MS medium for first 5 days was increased to two-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growth regulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemum germplasm.

Background : Ginseng seeds are one of short-lived seeds species which loose their viability easily in the condition of conventional storage. Cryopreservation using liquid nitrogen (LN) has been recommended as a alternative storage for this kind of germplasm short lived or dessiccation-sensitive. This study was performed to find out whether cryopreservation could assure initial viability not only for seeds with high germination rate but also for seeds with low germination rate (in aging process). Methods and Results : In this work, 3 cultivars of dehisced ginseng seeds were artificial aged in the condition of 40℃, 95% RH during 6 hours with 3 hours-interval. The germination rate of ginseng seeds was decreased to the range of 71 - 94% of initial viability by artificial aging treatment. After 24 hours of vapor-LN exposure on both of artificial aged and non treated seeds as a cryopreservation, germination rate for each cultivar was decreased with the range of 76 - 95% of initial viability. While the decreasing patterns of germination rate for each cultivar showed similar curves between before and after vapor-LN exposure, the aging effects could be slightly little by cryopreservation for 3 cultivars of ginseng seeds. Conclusion : From the above results, we may suggest that cryopreservation could be recommened for storage tool of dehisced ginseng seeds even with low viability also and expected to make slower seed aging process during preservation period through further study.