Experimental results concerning the effect of sucrose concentration on cell biomass and synthesized anthocyanin amount in the callus culture

Volume 16(1), 91-99, 2012 JOURNAL of Horticulture, Forestry and Biotechnology www.journal-hfb.usab-tm.ro Experimental results concerning the effect of sucrose concentration on cell biomass and synthesized anthocyanin amount in the callus culture Lazăr A. 1*, Petolescu Cerasela 1 1 Banat s University of Agricultural Sciences and Veterinary Medicine Timisoara, Faculty of Horticulture and Forestry *Corresponding author. Email: alexlazar2002@yahoo.com Abstract The growth rate of cell biomass registered maximum mean value in the cultivation interval of 24 days for Burgund Mare, Cabernet Sauvignon and Pinot Noir and 27 days for Negru Tinctorial, Oporto and Cabernet Sauvignon varieties. Regarding the sucrose concentration added into the culture medium, it has been observed that callus growth rate registered for the concentration of 30 g/l and the most reduced values for callus growth rate were registered for the sucrose concentration of 60 g/l. Comparing the mean obtained values for the studied grapevine varieties, it has been observed that the largest values for synthesized anthocyanins in callus culture has been registered for Negru Tinctorial variety followed by Burgund Mare, Oporto, Merlot, Pinot Noir and Cabernet Sauvignon. The same hierarchy is maintained for behaviour of grapevine varieties in terms of each type of sucrose concentration added into culture media. The differences found between varieties depending on anthocyanin amount from callus cultures were very significant in most cases. Key words callus culture, cell biomass, anthocyanin, Vitis vinifera L. The in vitro cell cultures depends on a number of factors of which we mention the kind of explants, the growing medium, genotype and hormone balance. The quantity of cell biomass in cell cultures callus depends by choosing the most appropriate factors. One element that allows assessment of the potential performance of obtaining cell biomass is sucrose concentration. Anthocyanins are natural pigments that demonstrate homeostatic, hypotensive, cardioprotective and anti-tumor properties (Markakis, P. [7], Rao, S.R., Ravishankar, G.A.[11]). In Romania, it is found a large diversity of vegetal sources that may serve as raw materials for anthocynin extraction among which one that proves very potential is Vitis genus (Lazăr, A., Nedelea G.[3,4,5], Tărăbăşanu-Mihailă, C.N.[12]). The research initiated for the six grapevine (Burgund Mare, Cabernet Sauvignon, Merlot, Oporto, Negru Tinctorial and Pinot Noir) varieties consists in obtaining of anthocyanins (Lofty S. [6], Yamakawa T. [13]) from cell biomass (Gamborg, O.L. [1], Lazăr, A. [2,5], Murashige, T., Skoog, F. [8], Oluf, L.C. [9],. Payne, G. F. [10]) after 6, 12, 18, 24, 30 and 36 days of cultivation. In the present paper, I will present only the results obtaining after 30 days of cell culture. Biological Material and Methods The biological material, donor of explants consisted of six grapevine varieties found in the Ampelographic collection of Faculty of Horticulture and Forestry of Banat s University of Agricultural Science Timişoara. The studied grapevine varieties for red wine are Burgund Mare, Cabernet Sauvignon, Oporto, Merlot, Negru Tinctorial and Pinot Noir. The culture medium that have generated best results was M6, these containing a hormone balance including an auxin, α-naphthalene acetic acid and a cytokinin, kinetin. M6 culture medium represents a variant of Murashige-Skoog (Gamborg, O.L. [1], Lazăr, A. [2, 4, 5] medium supplemented with auxin and cytokinin in a ration 4:1. The research initiated for the six grapevine varieties consists in obtaining of cell biomass via callus cultures using a 20, 30, 50 and 60 g/l sucrose concentration in the culture medium and the chemical extraction from the cell biomass and quantitative determination of anthocyanin fractions using spectrophotometry. The anthocyanin compounds were extracted from cell cultures using acidulated methanol solution 1% chloride acid. The extraction has been left to rest over night at 4 C. 91

After extraction, the samples were centrifuged for 3 minutes at 5000 rot/minute and then the supernatant was analyzed using the spectrophotometer at variable wave length UV-VIZ JENWAY 6505. The UV-VIZ spectra were traced for the wave length domain 350 700 nm, considering all studied grapevine varieties and the wave length for which the absorbance was maximum (λ = 525 nm) was identified. The total anthocyanins were determined being expressed as cyanidin- 3 glucoside using the molar extinction coefficient 2,95 x 10 4. Results and Discussions Analyzing the studied grapevine varieties, it has been observed that the growth rate of cell biomass increases proportionally with cultivation period reaching maximum mean values of 250,75% for Negru Tinctorial after 27 days followed by Oporto, Merlot, Pinot Noir, Cabernet Sauvignon and Burgund Mare variety with mean values of 162,11%, the difference between this and Negru Tinctorial being of 88,64% (Table 1). using callus culture on Burgund Mare variety duration 30 20 50 60 (days) 3 100,50 100,24 100,47 100,20 100,35 0,35-6 103,96 113,44 101,52 110,81 107,43 7,43-9 127,99 133,28 101,60 113,03 118,98 18,98-12 173,01 175,67 102,47 113,57 141,18 41,18 * 15 224,75 224,75 123,34 135,46 177,08 77,08 *** 18 244,70 243,70 149,77 170,95 202,28 102,28 *** 21 295,88 281,42 186,25 193,79 239,34 139,34 *** 24 303,23 307,44 219,27 218,50 262,11 162,11 *** 27 302,49 302,51 218,72 213,23 259,24 159,24 *** 30 295,86 293,15 215,06 210,48 253,64 153,64 *** 33 284,32 284,19 209,28 203,83 245,40 145,40 *** 36 263,99 279,47 208,14 202,04 238,41 138,41 *** Average 246,80 247,79 173,29 176,71 DL 5% =32,21 DL 1% =43,14 Val. rel. (%) 100,00 100,40 70,21 71,60 DL 01% =56,89 Differences 0,00 1,00-73,51-70,09 DL5%=16,13 DL1%=21,80 DL01%=29,04 using callus culture on Negru Tinctorial variety duration 30 20 50 60 (days) 3 100,53 100,43 100,88 100,77 100,65 0,65-6 101,16 100,86 102,43 101,15 101,40 1,40-9 121,79 122,03 102,50 101,02 111,83 11,83-12 179,72 176,56 103,26 101,90 140,36 40,36-15 231,38 226,02 137,44 108,30 175,78 75,78 ** 18 269,32 256,35 158,85 128,72 203,31 103,31 *** 21 282,19 265,67 182,74 145,91 219,13 119,13 *** 24 298,84 281,96 202,76 163,99 236,89 136,89 *** 27 316,87 301,31 223,54 161,27 250,75 150,75 *** 30 320,99 298,04 219,30 150,48 247,20 147,20 *** 33 318,93 291,54 198,58 144,74 238,44 138,44 *** 36 295,89 285,67 204,86 130,14 229,14 129,14 *** Average 258,60 245,65 173,26 133,60 DL 5% =47,88 DL 1% =64,12 Val. rel. (%) 100,00 94,99 67,00 51,66 DL 01% =84,57 Differences 0,00-12,95-85,34-125,00 DL 5% =24,48 DL 1% =33,08 DL 01% =44,06 Table 1 Table 2 92

Table 3 using callus culture on Oporto variety duration (days) 30 20 50 60 3 100,66 100,64 100,84 101,21 100,84 0,84-6 111,23 109,03 103,04 101,61 106,23 6,23-9 121,96 122,95 103,40 102,14 112,61 12,61-12 155,27 142,30 103,99 103,69 126,31 26,31-15 185,71 172,67 120,91 116,31 148,90 48,90 *** 18 217,01 216,70 150,90 144,47 182,27 82,27 *** 21 235,74 238,03 177,98 173,36 206,28 106,28 *** 24 266,24 254,66 201,81 198,18 230,22 130,22 *** 27 288,56 270,26 200,49 196,23 238,89 138,89 *** 30 283,35 264,34 196,90 195,61 235,05 135,05 *** 33 274,31 263,69 193,43 195,22 231,66 131,66 *** 36 269,99 261,28 189,27 193,76 228,58 128,58 *** Average 226,55 217,91 163,73 161,75 DL 5% =25,93 DL 1% =34,72 Val. rel. (%) 100,00 96,19 72,27 71,40 DL 01% =45,79 Differences 0,00-8,64-62,81-64,80 DL5%=13,06 DL1%=17,65 DL01%=23,51 The growth rate of cell biomass registered maximum mean value in the cultivation interval of 24 days for Burgund Mare, Cabernet Sauvignon and Pinot Noir and 27 days for Negru Tinctorial (Table 2), Oporto (Table 3) and Cabernet Sauvignon varieties. Moreover, the growth rate for all studied grapevine varieties did not show a significant increase in the first 9 to 12 cultivation days, value that become significant beginning with the 12th day for Burgund Mare, Merlot (Table 4), Pinot Noir and later on the 15th day for the remaining varieties. Regarding the sucrose concentration added into the culture medium, it has been observed that callus growth rate registered for the concentration of 30 g/l, values comprised between 258,60% for Negru Tinctorial and 183,62% for Cabernet Sauvignon (Table 6), the difference between these being of 74,98% (Lazăr, A. [3]). The most reduced values for callus growth rate were registered for the sucrose concentration of 60 g/l, the values being between 161,75% for Pinot Noir and 131,53% for Cabernet Sauvignon, the difference between these being of 30,22%. Burgund Mare and Merlot registered the lowest values for the sucrose concentration of 50 g/l. Table 4 using callus culture on Merlot variety duration (days) 30 20 50 60 3 101,16 100,67 101,91 100,94 101,17 1,17-6 110,30 105,80 105,60 106,46 107,04 7,04-9 118,95 118,61 106,94 107,83 113,08 13,08-12 143,42 141,92 120,35 115,52 130,30 30,30 * 15 188,69 170,77 126,39 132,97 154,71 54,71 *** 18 210,17 206,08 139,53 166,86 180,66 80,66 *** 21 253,11 245,09 169,71 185,49 213,35 113,35 *** 24 269,91 263,37 195,46 193,13 230,47 130,47 *** 27 267,03 262,69 194,78 191,26 228,94 128,94 *** 30 265,71 252,78 192,38 190,80 225,42 125,42 *** 33 250,42 247,77 187,54 190,09 218,95 118,95 *** 36 249,18 242,76 188,06 189,63 217,41 117,41 *** Average 219,11 212,48 161,12 165,56 DL 5% =25,27 DL 1% =33,85 Val. rel. (%) 100,00 96,97 73,53 75,56 DL 01% =44,64 Differences 0,00-6,63-57,99-53,56 DL 5% =13,53 DL 1% =18,28 DL 01% =24,35 93

In most of studied grapevine varieties, expect for Burgund Mare where callus was induced on a sucrose concentration of 20 g/l and registered the highest value of cell growth, the callus induced on media with 30 g/l sucrose concentration have generated the largest values for cell growth. Table 5 using callus culture on Pinot Noir variety duration (days) 30 20 50 60 3 101,49 101,70 100,79 100,69 101,17 1,17-6 103,16 104,38 101,63 101,90 102,77 2,77-9 125,14 131,39 101,15 101,54 114,80 14,80-12 157,15 145,63 102,84 102,57 127,05 27,05 * 15 181,54 167,41 116,03 119,57 146,14 46,14 *** 18 230,53 198,52 142,06 152,89 181,00 81,00 *** 21 244,79 236,07 170,81 178,58 207,57 107,57 *** 24 254,38 248,79 196,02 195,41 223,65 123,65 *** 27 251,96 245,00 193,70 185,75 219,10 119,10 *** 30 246,72 243,10 193,76 184,23 216,95 116,95 *** 33 231,10 224,52 190,49 181,95 207,01 107,01 *** 36 219,86 216,63 181,25 171,37 197,28 97,28 *** Average 210.43 202.16 158.73 157.31 DL 5% =21,56 DL 1% =28,88 Val. rel. (%) 100,00 96.07 75.43 74.76 DL 01% =38,09 Differences 0,00-8.27-51.70-53.12 DL5%=11,12 DL1%=15,03 DL01%=20,02 Comparatively with the concentration of 30 g/l used as control variant, the sucrose concentrations of 50 g/l and 60 g/l have registered growth rates significantly inferior. The sucrose concentration of 20 g/l has generated values of cell growth rate close to control variant and without significant differences. Table 6 using callus culture on Cabernet Sauvignon variety duration (days) 30 20 50 60 3 100,91 100,91 100,16 100,47 100,61 0,61-6 102,70 104,93 100,97 101,26 102,46 2,46-9 113,26 117,78 101,75 104,36 109,29 9,29-12 132,42 144,07 103,89 106,73 121,78 21,78-15 160,01 158,21 109,72 109,62 134,39 34,39 ** 18 178,17 184,51 125,54 118,74 151,74 51,74 *** 21 199,05 200,93 149,65 142,57 173,05 73,05 *** 24 211,10 210,85 152,05 150,86 181,21 81,21 *** 27 218,13 210,57 151,25 150,30 182,56 82,56 *** 30 216,63 208,82 150,24 149,56 181,31 81,31 *** 33 215,38 207,94 149,22 144,36 179,22 79,22 *** 36 213,05 205,55 149,10 142,87 177,64 77,64 *** Average 183,62 182,12 134,02 131,53 DL 5% =22,56 DL 1% =30,21 Val. rel. (%) 100,00 99,19 72,99 71,63 DL 01% =39,84 Differences 0,00-1,49-49,60-52,09 DL5%=11,48 DL1%=15,51 DL01%=20,67 94

The effect of sucrose concentration on total anthocyanin synthesis (mg/g callus) after 30 days in case of studied grape varieties using callus culture Sucrose 20 g/l 30 g/l 50 g/l Average Grapevine variety x s s x % sx x s % x s s x % x s s x % Merlot 3,30+0,06 3,01 3,51+0,04 1,99 3,50+0,05 2,66 3,43+0,07 3,44 Table 7 Negru Tinctorial 11,56+0,08 1,24 12,48+0,03 0,37 12,71+0,10 1,33 12,25+0,35 4,95 Pinot Noir 2,77+0,15 9,37 2,89+0,01 0,68 2,88+0,04 2,13 2,85+0,04 2,48 Burgund Mare 6,11+0,01 0,39 6,13+0,07 2,04 5,91+0,07 2,13 6,05+0,07 2,01 Oporto 5,39+0,03 0,89 5,40+0,06 1,99 5,44+0,08 2,66 5,41+0,02 0,50 Cabernet Sauvignon 1,03+0,06 10,79 1,30+0,02 3,03 1,31+0,03 3,33 1,21+0,09 12,82 Average 5,03+1,51 73,39 5,28+1,60 74,39 5,29+1,63 75,71 5,20+0,08 15,13 The comparison of variety mean regarding the synthesized anthocyanins (mg/g callus) after 30 days using callus culture Grapevine varieties Negru T Merlot 12,25 3,43 356,69 8,82*** Pinot N Merlot 2,85 3,43 82,91-0,59000 Burgund M Merlot 6,05 3,43 176,18 2,62*** Oporto Merlot 5,41 3,43 157,63 1,98*** Cabernet S Merlot 1,21 3,43 35,34-2,22000 Pinot N Negru T 2,85 12,25 23,25-9,40000 Burgund M Negru T 6,05 12,25 49,39-6,20000 Oporto Negru T 5,41 12,25 44,19-6,84000 Cabernet S Negru T 1,21 12,25 9,91-11,04000 Burgund M Pinot N 6,05 2,85 212,48 3,20*** Oporto Pinot N 5,41 2,85 190,11 2,57*** Cabernet S Pinot N 1,21 2,85 42,62-1,63000 Oporto Burgund M 5,41 6,05 89,47-0,64000 Cabernet S Burgund M 1,21 6,05 20,06-4,84000 Cabernet S Oporto 1,21 5,41 22,42-4,20000 DL5%=0,10 DL1%=0,13 DL0,1%=0,17 Table 8 The research initiated for the six grapevine varieties consists in quantitative determination of anthocyanin fractions from the cell biomass via callus cultures obtained on 20, 30 and 50 g/l sucrose concentration (Table 7). The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) after 30 days using callus culture 30 g/l 20 g/l 5,28 5,03 105,14 0,26*** 50 g/l 20g /l 5,29 5,03 105,28 0,27*** 50 g/l 30 g/l 5,29 5,28 100,14 0,01 DL5%=0,07 DL1%=0,11 DL0,1%=0,21 Table 9 Considering all studied varieties from the view point of synthesized anthocyanin amount in cell culture, it is 95

clearly observed that the highest values were obtained in callus cultures for Negru Tinctorial variety (Table 8). In case of Negru Tinctorial variety, the registered values were situated in the interval 10,04 12,48 mg/g in callus cultivation conditions with 20 g/l sucrose concentration and 30 g/l, respectively, the variation amplitude being of 2,44 mg/g. The values were registered after 6 days and 30 days, respectively. The most reduced values of anthocyanins were registered for Cabernet Sauvignon variety with values comprised in the interval 0,70 1,31 mg/g and variation amplitude of 0,61 mg/g (table 4). These values were obtained in callus cultivation conditions on culture media with 30 g/l and 50 g/l sucrose (Table 9), respectively, the values being measured after 6 and 30 cultivation days. Comparing the mean obtained values for the studied grapevine varieties, it has been observed that the largest values for synthesized anthocyanins in callus culture has been registered for Negru Tinctorial variety followed by Burgund Mare, Oporto, Merlot, Pinot Noir and Cabernet Sauvignon. Table 10 The comparison of studied grape varieties regarding the synthesized anthocyanins (mg/g callus) a) The comparison of studied grape varieties regarding the synthesized anthocyanins (mg/g callus) using 20 g/l sucrose Grapevine varieties Negru T Merlot 11,56 3,30 350,58 8,26*** Pinot N Merlot 2,77 3,30 83,88-0,53000 Burgund M Merlot 6,11 3,30 185,18 2,81*** Oporto Merlot 5,39 3,30 163,56 2,10*** Cabernet S Merlot 1,03 3,30 31,36-2,26000 Pinot N Negru T 2,77 11,56 23,93-8,80000 Burgund M Negru T 6,11 11,56 52,82-5,45000 Oporto Negru T 5,39 11,56 46,65-6,17000 Cabernet S Negru T 1,03 11,56 8,95-10,53000 Burgund M Pinot N 6,11 2,77 220,77 3,34*** Oporto Pinot N 5,39 2,77 194,99 2,63*** Cabernet S Pinot N 1,03 2,77 37,39-1,73000 Oporto Burgund M 5,39 6,11 88,32-0,71000 Cabernet S Burgund M 1,03 6,11 16,94-5,07000 Cabernet S Oporto 1,03 5,39 19,18-4,36000 b) The comparison of studied grape varieties regarding the synthesized anthocyanins (mg/g callus) using 30 g/l sucrose Grapevine varieties Negru T Merlot 12,48 3,51 355,86 8,97*** Pinot N Merlot 2,89 3,51 82,49-0,61000 Burgund M Merlot 6,13 3,51 174,90 2,63*** Oporto Merlot 5,40 3,51 154,05 1,90*** Cabernet S Merlot 1,30 3,51 36,97-2,21000 Pinot N Negru T 2,89 12,48 23,18-9,59000 Burgund M Negru T 6,13 12,48 49,15-6,35000 Oporto Negru T 5,40 12,48 43,29-7,08000 Cabernet S Negru T 1,30 12,48 10,39-11,18000 Burgund M Pinot N 6,13 2,89 212,02 3,24*** Oporto Pinot N 5,40 2,89 186,74 2,51*** Cabernet S Pinot N 1,30 2,89 44,81-1,60000 Oporto Burgund M 5,40 6,13 88,08-0,73000 Cabernet S Burgund M 1,30 6,13 21,13-4,84000 Cabernet S Oporto 1,30 5,40 24,00-4,11000 96

c) The comparison of studied grape varieties regarding the synthesized anthocyanins (mg/g callus) using 50 g/l sucrose Grapevine varieties Negru T Merlot 12,71 3,50 363,27 9,21*** Pinot N Merlot 2,88 3,50 82,43-0,61000 Burgund M Merlot 5,91 3,50 168,97 2,41*** Oporto Merlot 5,44 3,50 155,63 1,95*** Cabernet S Merlot 1,31 3,50 37,46-2,19000 Pinot N Negru T 2,88 12,71 22,69-9,82000 Burgund M Negru T 5,91 12,71 46,51-6,80000 Oporto Negru T 5,44 12,71 42,84-7,26000 Cabernet S Negru T 1,31 12,71 10,31-11,40000 Burgund M Pinot N 5,91 2,88 204,98 3,03*** Oporto Pinot N 5,44 2,88 188,81 2,56*** Cabernet S Pinot N 1,31 2,88 45,45-1,57000 Oporto Burgund M 5,44 5,91 92,11-0,47000 Cabernet S Burgund M 1,31 5,91 22,17-4,60000 Cabernet S Oporto 1,31 5,44 24,07-4,13000 DL5%=0,17 DL1%=0,23 DL0,1%=0,30 Table 11 The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) a) The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) on Burgund Mare variety 30 g/l 20 g/l 6,13 6,11 100,43 0,03 50 g/l 20g /l 5,91 6,11 96,78-0,200 50 g/l 30 g/l 5,91 6,13 96,37-0,220 b) The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) on Cabernet Sauvignon variety 30 g/l 20 g/l 1,30 1,03 125,33 0,26** 50 g/l 20g /l 1,31 1,03 126,71 0,28** 50 g/l 30 g/l 1,31 1,30 101,10 0,01 c) The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) on Merlot variety 30 g/l 20 g/l 3,51 3,30 106,34 0,21* 50 g/l 20g /l 3,50 3,30 106,07 0,20* 50 g/l 30 g/l 3,50 3,51 99,75-0,01 d) The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) on Oporto variety 30 g/l 20 g/l 5,40 5,39 100,16 0,01 50 g/l 20g /l 5,44 5,39 100,93 0,05 50 g/l 30 g/l 5,44 5,40 100,78 0,04 97

e) The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) on Negru Tinctorial variety 30 g/l 20 g/l 12,48 11,56 107,94 0,92*** 50 g/l 20g /l 12,71 11,56 109,91 1,15*** 50 g/l 30 g/l 12,71 12,48 101,83 0,23** f) The comparison of sucrose concentrations regarding the synthesized anthocyanins (mg/g callus) on Pinot Noir variety 30 g/l 20 g/l 2,89 2,77 104,58 0,13 50 g/l 20g /l 2,88 2,77 104,24 0,12 50 g/l 30 g/l 2,88 2,89 99,67-0,01 The same hierarchy is maintained for behaviour of grapevine varieties in terms of each type of sucrose concentration added into culture media. The differences found between varieties depending on anthocyanin amount from callus cultures were very significant in most cases. Comparing the sucrose concentrations in terms of synthesized anthocyanins, it has been observed that in most cases, the highest values of anthocyanins were registered in case of culture media with concentration of 30 g/l sucrose. The most reduced values were registered for sucrose concentrations of 20 g/l (Table 11). The differences between sucrose concentrations regarding the anthocyanin amount are in most cases statistically covered. Conclusions The sucrose concentration added into culture medium influenced directly the callus growth. In case of Negru Tinctorial, Cabernet Sauvignon, Pinot Noir, Merlot and Oporto varieties the optimal concentration was of 30 g/l, while for Burgund Mare 20 g/l. The concentrations of 50 and 60 mg/l proved to generate an inhibitory effect on growth rate. The study results concerning the influence of sucrose concentration on biosynthetic potential of calli revealed statistically insured differences between anthocyanin yield and sucrose concentration added into culture medium. The supplementation of culture media for calli with 30g/l generated a positive effect on their biosynthetic potential. On contrary, the reduction of sucrose amount to 20g/l determined implicitly a reduction of anthocyanin amount synthesized by calli. The differences registered between biosynthetic potential of calli rising from different varieties on culture media with the same sucrose amount, were very significant which indicates the major role of genetic component on in vitro anthocyanins production. References 1. Gamborg, O.L., Murashige, T., Thorpe, T.A., Vasil, I.K., 1976 - Plant tissue culture media, In Vitro, 7, p. 473-478. 2. Lazăr, A., Lăpădătescu Simona, Nedelea G., 2004 - Studies concerning in vitro reactivity of five Vitis vinifera L. cultivars, Cercetări ştiinţifice seria a VIII-a, Biotehnologie şi Biodiversitate, Facultatea de Horticultură, Timişoara, p.49-52 3. Lazăr, A., Nedelea G., 2007 - Studies concerning in vitro response of some red grape varieties (Vitis vinifera L.), Lucrări Ştiinţifice vol. 15 (2), Realizări şi perspective în horticultură, viticultură, vinificaţie şi silvicultură, Chişinău, p.79-82. 4. Lazăr, A., Nedelea G., 2007 - Researches concerning the determination of the anthocyanin fractions from cell cultures of Vitis vinifera L. using high performance liquid chromatography (HPLC), Lucrări Ştiinţifice vol. 15 (2), Realizări şi perspective în horticultură, viticultură, vinificaţie şi silvicultură Chişinău, p.104-106. 5. Lazăr, A., 2008 - Researches concerning the obtaining of some secondary metabolites using in vitro cultures in Vitis vinifera L., Teza de doctorat, Timişoara, p.247-254. 6. Lofty S., Fleuriet A., Ramos T., Macheix J.J., 1989 - Biosynthesis of phenolic compounds in Vitis vinifera cell suspension cultures: Study of hydroxycimamoyl CoA: ligase, Plant Cell Reports 8: p.93-96. 7. Markakis, P., 1982 - Antocyanins as Food Colours New York: Academic Press, p 254. 8. Murashige, T., Skoog, F., 1962 - A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant., 15, p.473-497. 9. Oluf, L.C., Jerry, P.S., 1981 - Nutrition, Media and Characteristics of Plant Cell and Tissue cultures, Plant Tissue, Ed. Academic Press, p. 21-45. 98

10. Payne, G. F., Bringi, V., Prince, C., Shuler, M. L., 1991 - Suspension culture -Plant Cell and Tissue in Liquid Systems, Hanser, p. 145-176. 11. Rao, S.R., Ravishankar, G.A., 2002 - Plant cell cultures: Chemical factories of secondary metabolites, Biotech. Advances 20, p.101-153 12. Tărăbăşanu-Mihailă, C.N., Gorduza, V.M., Radu, F., Măzgăreanu, M., 1997 -Coloranţi organici de interes alimentar, cosmetic şi farmaceutic Bucureşti: Uni-Press, p.331 13. Yamakawa T., Kato S., Ishida K., Kodama T., Minoda Y., 1983 - Formation and identification of anthocyanins in cultured cell of Vitis sp., Agr. Biol. Chem., 47(5), 997-1001. Acknowledgement This work was published during the project POSTDOCTORAL SCHOOL OF AGRICULTURE AND VETERINARY MEDICINE", POSDRU/89/1.5/S/62371, co-financed by the European Social Fund through the Sectorial Operational Programme for the Human Resources Development 2007-2013. 99