This study was conducted to improve and supplement the system of cryopreservation for adventitious bulbs induced by tissue cultured bulb-scales of lily (Lilium spp.) cvs. ‘Milky way’. The explants, bulblets and bulb-scale-bulblets, were treated to low temperature (4℃) for 7 days prior to the pre-culture. The adventitious bulbs were pre-cultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3 and 0.7M). The pre-cultured adventitious bulbs were treated to loading solution (LS1 or LS2, C4 or C6) containing 35% of PVS3 (LS1, C4) or 40% of PVS3 (LS2, C6) for 40 min and exposed to dehydration solution (PVS3, B1) containing 50% glycerol and 50% sucrose for 60 min at 25℃. The adventitious bulbs were moved onto droplets containing 3 μl PVS3 on sterilized aluminum foils, and then soaked into liquid nitrogen (LN) for 60 min. The result of highest regrowth rate as 65.7% was obtained in cold treatment (4℃), osmoprotected with LS1 solution, and cultured in PCM3 medium by using bulb-scale-bulblet for cryopreservation. This result shows that droplet-vitrification could be used as a promising method for long-term storage of lily genetic resource.

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.

We compared the germination rate of dehisced ginseng (Panax ginseng) seeds that were dried under two different conditions, slowly at 15℃ [relative humidity (RH) 10-12%] and rapidly under a laminar airflow cabinet at 25℃ (RH 22-25%). The measurements showed that drying rate and seed moisture content (SMC) play important roles in storage ability and vigor. The seeds that were dried rapidly at 25℃ showed high GR compared with the seeds that were dried at 15℃ after 6 and 12 months of storage at -80℃ irrespective of MC. Seeds dried slowly at 15℃ with MC higher than 7.0% showed high GR maintenance after storage at -18℃ and at 4℃ in comparison with rapidly dried seeds. However, the GR of the slowly desiccated seeds decreased as mean SMC was reduced to less than 5.0%, whereas the rapidly dried seeds were distinguished by significantly high GR irrespective of the storage conditions. The ginseng seeds desiccated under different conditions showed differences in storage performance. Seeds with 7-9% MC that were dried slowly at 15℃ for 5-7 days showed high GR after 4℃ and -18℃ storage; however, longer periods of desiccation decreased the germination level remarkably compared with that of rapidly dried seeds.

Potato virus Y (PVY) and potato leafroll virus (PLRV) are among the most damaging potato viruses and prevalent in most potato growing areas. In this study, cryopreservation was used to eradicate PVY and PLRV using two cryogenic methods. Potato shoot tips proliferated in vitro were cryopreserved through droplet-vitrification and encapsulation-vitrification using plant vitrification solution 2 (PVS2; 30% glycerol + 15% dimethyl sulfoxide + 15.0% ethylene glycol + 13.7% sucrose) and modified PVS2. Both cryogenic procedures produced similar rates of survival and regrowth, which were lower than those from shoot tip culture alone. The health status of plantlets regenerated from shoot tip culture alone and cryopreservation was checked by reverse transcription-polymerase chain reaction. The frequency of virus-free plants regenerated directly from highly proliferating shoot tips reached 42.3% and 48.6% for PVY and PLRV, respectively. In comparison, the frequency of PVY and PLRV eradication after cryopreservation was 91.3~99.7% following shoot-tip culture. The highest cryopreserved shoot tip regeneration rate was observed when shoot tips were 1.0~1.5 mm in length, but virus eradication rates were very similar (96.4~99.7%), regardless of shoot tip size. This efficient cryotherapy protocol developed to eliminate viruses can also be used to prepare potato material for safe long-term preservation and the production of virus-free plants.

It is very crucial to evaluate the genetic diversity of peanut genetic resources for identification of peanut germplasm accessions and variety improvement. Cultivated peanut generally has two subspecies, hypogaea and fastigiata. In this study, we identified peanut into three plant types, virginia (var. hypogaea), spanish (var. vulgaris), and valencia (var. fastigiata). Former one belongs to ssp. hypogaea and latter two are involved in ssp. fastigiata. Twenty SSR markers were used to assess the genetic variation of three sets, hypogaea, vulgaris, and fastigiata, respectively. Out of variety-specific SSR primers tried in this study, ten pairs of SSR primers showed polymorphisms. Each accession could be identified by a specific set of polymorphic SSR primers, and allele number was evaluated among accessions, with an average of 6.7 in var. hypogaea and 5.4 in var. vulgaris and fastigiata. For evaluation of genetic diversity, gene diversity ranged from 0.336 to 0.844 and PIC (polymorphism information contents) ranged from 0.324 to 0.827 were investigated. Dendrograms based on genetic distances were constructed, which showed the existence of three different clusters. And these three different clusters might be associated with the genes involved in three plant types. The results also suggested that there were plentiful SSR polymorphisms among peanut germplasm accessions in RDA (Rural Development Administration, Korea) Genebank and SSRs might play an important role in evaluating peanut accessions and cultivar improvement.