Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l^–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.     

Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region

Dihydroflavonol reductase (DFR) is a key enzyme involved in anthocyanin biosynthesis and proanthocyanidin synthesis in grape. DFR catalyses the reduction of dihydroflavonols to leucoanthocyanidins in the anthocyanin pathway. The DFR products, the leucoanthocyanidins, are substrates for the next step in the anthocyanin pathway and are also the substrates for the proanthocyanidin pathway. In the present study the promoter of the grape dfr gene was cloned. Analysis of the dfr promoter sequence revealed the existence of several putative DNA binding motifs. The dfr promoter was fused to the uidA gene and the control of this fusion and the endogenous dfr gene expression, was studied in transformed plants and in red cell suspension originated from fruits. The dfr promoter-uidA gene fusion was expressed in leaves, roots and stems. Deletions of the dfr promoter influenced the specificity of the expression of the GUS gene fusion in plantlet roots and the level of expression in plants and in the red cell suspension originated from fruits. The deletion analysis of the dfr promoter suggests that a specific sequence located between -725 to -233 might be involved in expression of the dfr gene in fruits. Light, calcium and sucrose induced the dfr gene expression. In the transformed suspension cultures, expression of both the endogenous dfr gene and the dfr promoter-uidA gene fusions was induced by white light. The induction by both light and calcium suggests the possible involvement of a UV receptors signal transduction pathway in the induction of the dfr gene. The induction of the dfr gene and the dfr promoter-uidA gene fusions by light and sucrose indicates a close interaction between sucrose and light signalling pathways.