Scientific Papers Series B Horticulture

The characterization of the grapevine varietiesbased on free amino acid profile shows a greatimportance to complement the modern methodson investigation of the vine varieties(isoenzymatic analysis and DNA analysis).MATERIALS AND METHODSThe biological material was represented by fivelocal varieties to Galben de Odobetisortogroup (Galben de Odobeti, Zghihar deHui, Btut neagr, Negru moale and Negruvârtos), belonging ampelographic collection ofResearch and Development Station forViticulture and Winemaking Odobeti.Samples for analyses were the young leafobtained by forcing cuttings - eye. The leafsamples (2 or 3 leaves, ~ 5 g ) were collectedfrom each variety in plastic bags, labeled,stored on ice and brought to laboratory foranalyses.For extraction of free amino acids from theleaves of the vine has been used the methodBieleski & Turner (1966) adjusted forgrapevine. This method ensures the extraction,separation and quantitative estimation of aminoacids from small samples of vegetal freshtissue, by thin layer chromatography technique.For extraction of free amino acids in biologicalmaterial and for the preparation of standardsolutions of amino acids was used a mixturesolution with methanol, chloroform and waterin a ratio of 12:5:3 v/v/v. For each grapevariety was weighed a sample of 0.5 gbiological material. The fresh leaves werelyophilized and crushed in a mortar with liquidnitrogen until was obtained a fine powder.0,200 g was transferred for each sample in aEppendorf tube 1.5 ml. Over powder was added200 ml extraction solution (methanol:chloroform: water - M/C/W) in ratio of 12/5/3(v/v/v), which allowed the removal of pigmentsand lipids from plant material in chloroformlayer. Cell suspensions were treated with sonicfrequencies for 1 hour at 4 ° C, causing rupturecell membranes. After sonication the sampleswere stored in a refrigerator at 4 º C for at least1 h. To obtain the supernatant containing theamino acids, the sample was centrifuged at13,500 rpm for 15 minutes at 4 ° C.The chromatographic separation of amino acidswas achieved on TLC plates with silica gel164stationary phase 60 on aluminum foil 20 x 20(TLC Silicagel 60), manufactured by MerckKGaA, Germany. TLC plates were made withsize 10 x 7 cm and have been marked the areasof application for samples. Using standardamino acids were produced by Merck KGaA,Germany, in the solution of methanol:chloroform: water (12:5:3 v / v / v). Samples (2l) were applied with an automaticmicropipette with volume control, from left toright at the bottom of the plate, in the areasmarked for each cultivar. The chromatographicseparation or development of plates was donein mono dimension, was used the developmentsystem based on n - butanol / glacial acetic acid/ water (ratio 4:1:1 v/v/v). Afterchromatographic separation, the TLC plates areready for viewing and identification of aminoacids separated. To ensure reproducible results,the tests were repeated three times forpredominant amino acids, to all five varieties.View amino acids was performed usingspecific reagents – ninhydrin (a solution inconcentration of 0,25%). Amino acids appearas separate spots colors. The TLC platesviewed ninhydrin solution, sensitive to lightwere scanned or photographed for thequalitative and quantitative evaluation of freeamino acids separated from each sample. Toidentify amino acids separated in samples wascalculated the retention factor (Rf) comparedwith each standard amino acid. Quantitativedetermination of free amino acids in thesamples was performed using image analysisprogram - ImageJ ver 1.46 r, and the valuesobtained were expressed in g free amino acidfor 1 mg fresh leaf tissue.The data obtained were processed statisticallyfor analysis of variance (ANOVA test) and hasbeen determined significance of differences forthe total free amino acids content and the ratiobetween the predominant amino acids (Studenttest). Analysis of variance and Student test wasperformed using the Microsoft Excel menuTools - Data Analysis.RESULTS AND DISCUSSIONS14 amino acids were identified. In Figure 1 and2 are presented TLC plates with amino acidsseparated and visualized with ninyidrin solutionfor the five cultivars studied. Standard aminoacids used: alanine (Ala) and histidine (His). In