Jasminka Karoglan Kontić

Cryopreservation of grapevine ( Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).

Recovery of Flavonoids from Grape Skins by Enzyme- Assisted Extraction

Abstract Grape skin contains large amounts of different flavonoids so it can be used for their recovery. Optimization of enzyme-assisted extraction of flavonoids was conducted using oenological enzyme preparations with respect to enzyme dosage, temperature, extraction time, pH, and enzyme preparation. Optimal conditions were obtained using enzyme preparation Lallzyme EX-V, at the temperature of 45°C, time of 3 h, pH 2.0, and enzyme dosage of 10.52 mg/g. The new optimized extraction method is less expensive, simple, accurate, and selective for the recovery of simple flavonoids. It is based on an environmentally-friendly extraction solvent which may provide a valuable alternative to conventional methods.

In vitro introduction of healthy and virus-infected genotypes of native Croatian grapevine cultivars

We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L−1 benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.

Whole genome amplification and microsatellite genotyping of herbarium DNA revealed the identity of an ancient grapevine cultivar

Reconstruction of the grapevine cultivation history has advanced tremendously during the last decade. Identification of grapevine cultivars by using microsatellite DNA markers has mostly become a routine. The parentage of several renowned grapevine cultivars, like Cabernet Sauvignon and Chardonnay, has been elucidated. However, the assembly of a complete grapevine genealogy is not yet possible because missing links might no longer be in cultivation or are even extinct. This problem could be overcome by analyzing ancient DNA from grapevine herbarium specimens and other historical remnants of once cultivated varieties. Here, we present the first successful genotyping of a grapevine herbarium specimen and the identification of the corresponding grapevine cultivar. Using a set of nine grapevine microsatellite markers, in combination with a whole genome amplification procedure, we found the 90-year-old Tribidrag herbarium specimen to display the same microsatellite profile as the popular American cultivar Zinfandel. This work, together with information from several historical documents, provides a new clue of Zinfandel cultivation in Croatia as early as the beginning of fifteenth century, under the native name Tribidrag. Moreover, it emphasizes substantial information potential of existing grapevine and other herbarium collections worldwide.

Multi-response optimisation of ultrasound-assisted extraction for recovery of flavonoids from red grape skins using response surface methodology

INTRODUCTION For the characterisation of grape cultivars, the profile and content of flavonoids are important because these compounds impact grape and wine quality. To determine the correct profile and content of flavonoids, the use of robust, sensitive and reliable methods is necessary. OBJECTIVE The object of this research is to develop a new ultrasound-assisted extraction (UAE) method for the recovery of flavonoids from grape skins using response surface methodology. METHOD Optimisation of UAE was performed using a complementary study combining a Box-Behnken experimental design with qualitative analysis by high-performance liquid chromatography. RESULTS Optimal extraction conditions were obtained using the extraction solvent composed of acetonitrile:water:formic acid (26:73:1, v/v/v) at an extraction temperature of 50 °C, an extraction time of 15 min in a single-extraction step and with a solid-to-solvent ratio of 1:80 g/mL. The calculated relative standard deviations for the optimal extraction method were very low, measuring less than 5%. CONCLUSIONS This study demonstrates that numerous factors have strong effects on the extraction efficiency, including the type of organic modifier and its percentage in the extraction solvent, the number of extraction steps, the solid-to-solvent ratio, the extraction time and temperature and, finally, the particular nature of analyte and their position within the grape skin cell.

Grapevine yellows affecting the Croatian indigenous grapevine cultivar Grk

Abstract - The grapevine cultivar Grk, a close relative of Crljenak kaštelanski/Zinfandel, is grown exclusively in southern Croatia. Grapevine yellows-like symptoms were observed on vines in the vineyards in Lumbarda (southern Croatia) and in propagated grapevines near Zadar and Zagreb. The majority of the detected phytoplasma isolates belonged to the 16SrI group. However, RFLP pattern and R16F2n/R2 fragment sequence assigned one isolate to the 16SrIII group. Thus far, on cv. Grk, phytoplasmas belonging to three different groups have been detected: 16SrI, 16SrIII, and 16SrXII, which was confirmed previously. Aside from the 16SrI, 16SrV and 16SrXII phytoplasma groups previously found on grapevines in Croatia, the finding of 16SrIII group, which is not common on grapevines in Europe, adds to the diversity of phytoplasmas in a very small geographic region.

Polyphenolic Composition of the Berry Skin of Six Fungus-Resistant Red Grapevine Varieties

Phenolic compound profiles were investigated by high-performance liquid chromatography-diode array detector in three vintages to assess genotypic variation in berry skins of six resistant grapevine varieties: “Cabernet Cortis,” “Chancellor,” “Léon Millot,” “Maréchal Foch,” “Monarch,” and “Regent.” The research has revealed a significant difference among the varieties in the total phenolic content, anthocyanins, flavanols, and flavan-3-ols. A clear differentiation of all varieties by means of the polyphenol profile using discriminant analyses has been achieved. “Regent” reached the significantly highest total polyphenol content, as well as total anthocyanin and total flavonol content. The polyphenol profile of “Chancellor” was quite different from all of the other studied varieties. Although being closely related, “Maréchal Foch” and “Léon Millot” showed qualitatively and quantitatively quite different polyphenol contents. No clear connection between the genetic origin of the varieties and their polyphenol profiles has been found.

Extraction Methods of Polyphenol From Grapes: Extractions of Grape Polyphenols

Abstract The grape is one of the most important horticulture crops in the world and a reliable source of polyphenolic compounds. Nowadays, there are many papers concerning the analysis and especially the extraction of polyphenolic compounds from various parts of the grape berry. Numerous extraction techniques are applied for the recovery of polyphenolic compounds from grape berries including solid-liquid extraction, ultrasound-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction, etc. For all of these techniques, there are numerous published methods, but there is no standardized one. Thus, the aim of this chapter is to unify the known data concerning the extraction methods for the recovery of polyphenols from grapes.