Transcriptional changes of gibberellin oxidase genes in grapevines with or without gibberellin application during inflorescence development.

The concept that gibberellin (GA) application on seeded grapevines induces seedlessness has been known for decades in viticulture. GA was applied to inflorescence clusters of seeded diploid grapevine cultivar 'Tamnara' (Vitis spp.) at 14 days before full bloom (DBF). Morphological and molecular effects of GA application were examined on the induction of parthenocarpic fruit development. With GA application, ovaries were enlarged and pollen tube growth was completely inhibited. Vitis GA oxidase enzymes, key determinants for GA level, were characterized through phylogenetic analysis with Arabidopsis GA oxidase enzymes. Five VvGA 20-oxidase (VvGA20ox), three VvGA 3-oxidase (VvGA3ox), and nine VvGA 2-oxidase (VvGA2ox) family proteins, and one VvGA methyltransferase (VvGAMT) and one Vitis cytochrome P450 714A1 proteins were identified, and their expression patterns were analyzed during inflorescence development from 14 DBF to 5 days after full bloom (DAF). VvGA2ox1, VvGA20ox3, and VvGA3ox2 were the most abundantly expressed genes in each gene family at 7, 5, and 2 DBF, respectively. Following GA application at 14 DBF inducing seedlessness, GA catabolic genes such as VvGAMT2, VvGA2ox3, and VvGA2ox4 were up-regulated at 12 DBF, full bloom, and 5 DAF, respectively. Conversely, most GA biosynthetic genes, VvGA20oxs and VvGA3oxs, were down-regulated at near full bloom, and the timing of their peak expression was changed. These results suggest that GA application at pre-bloom changes the GA biosynthesis into GA catabolic pathway at near full bloom by altering the transcription level and timing of GA oxidase genes during grapevine inflorescence development.

Desiccation sensitivity and cryopreservation of Korean ginseng seeds.

Korean ginseng germplasm is maintained as clonal germplasm since there is no practical method for long-term seed conservation. The aim of this study was to establish a cryopreservation protocol for Korean ginseng seeds. Desiccation of undehisced ginseng seeds to a moisture content (MC) of 7.1 % did not decrease their dehiscence and germination. After cryopreservation, the dehiscence percentage of desiccated seeds decreased for MC above 12.5%; it was 26% for 22.6% seed MC and nil for 41.9% seed MC. Germination percentage did not decrease significantly between 12.5-22.6% seed MC, while germination percentage of dehisced seeds decreased below 7.2% MC, reaching 25.8% at 3.8% MC. After cryopreservation, the germination percentage decreased from 90.5-92.9% at 8.3-10.6% MC to 84.8% at 12.5% MC. At MCs below 8.3%, germination rapidly decreased from 85.0% at 7.2% MC to 34.9% at 5.3% MC. Therefore, the hydration window for cryopreservation of ginseng seeds is around 8-11% MC. Undehisced Korean ginseng seeds were characterized by their high lipid and protein content (lipids, 42.6% FW; proteins, 41.0% FW). When using thermal analysis, during the cooling phase, exothermic ice crystallization peaks were observed with dehisced ginseng seeds above 13.5% MCs (3.3 J/g FW). A second crystallization peak was detected following ice crystallization peaks.

Cryopreservation of garlic germplasm collections using the droplet-vitrification technique.

The droplet-vitrification protocol was applied to unripe inflorescences of plants of two Korean garlic collections, Danyang and Mokpo, to establish a cryopreserved germplasm collection. Garlic unripe inflorescences of the 59 accessions harvested at Danyang showed a mean survival of 83.3% and regeneration of 73.5% after cryopreservation. Unripe inflorescences of accessions cryopreserved at sub-optimal developmental stages displayed lower survival and/or regeneration. Of these 59 accessions, 53 were cryopreserved and stored for long-term conservation. In the Mokpo collection, unripe inflorescences of 149 accessions were cryopreserved, displaying a mean survival of 79.9% and regeneration of 78.2%. Of these 149 accessions, 116 were cryopreserved and stored for the long-term. A total of 252 accessions of five clonal Allium species, including garlic, were cryopreserved using unripe inflorescences, cloves or bulbils, with a mean survival of 80.9% survival and regeneration of 77.0%, from which 221 accessions were stored in liquid nitrogen for long-term conservation. The real-time quantitative, reverse transcription (RT)-PCR assay of several garlic viruses showed that virus concentration was much lower in plantlets originating from cryopreserved material, compared to plantlets originating from preculture control and dehydration control samples. These results demonstrate that large-scale implementation of cryopreservation of Allium germplasm is feasible and that it can result in the regeneration of virus-free or little infected material. These findings will strongly facilitate the conservation and international exchange of Allium germplasm.

Cryopreservation of cultivated and wild potato varieties by droplet vitrification: effect of subculture of mother-plants and of preculture of shoot tips.

In this paper, we studied the effect of subculture of mother-plants and of preculture of shoot tips of two potato varieties (Dejima, cultivated and STN13, wild) cryopreserved using the droplet-vitrification technique. The subculture conditions (light intensity, aeration and planting density) significantly affected survival of both non-cryopreserved and cryopreserved shoot-tips in both varieties. The subculture duration and the position of the shoot tips on the axis of the in vitro plantlets had a significant (P<0.0001) effect on survival of cryopreserved shoot tips. The optimal subculture duration was 7 and 5 weeks and the optimal size of shoot tips was 1.5-2.0 and 1.0-1.5 mm for var. Dejima and STN13, respectively. Survival of cryopreserved shoot tips was influenced by the sucrose concentration in the preculture medium and the preculture duration. The highest survival of cryopreserved shoot tips was observed after preculture with 0.3 M sucrose for 8 h followed by 0.7 M sucrose for 18 h. These results indicate that the parameters of the subculture of mother-plants and of preculture of shoot tips should be carefully optimized, especially in the case of wild species.

Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure.

The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.