Experimental results concerning the synthesized anthocyanin amount in the Vitis vinifera L. suspension cell culture in the laboratory bioreactor

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1 Experimental results concerning the synthesized anthocyanin amount in the Vitis vinifera L. suspension cell culture in the laboratory bioreactor Lazar A. 1*, Petolescu Cerasela 1 1 USAMVB Timisoara, Faculty of Horticulture and Sylviculture *Corresponding author. alexlazar2002@yahoo.com Abstract: The researches approached have taken into consideration six varieties of grapevines and were aiming the chemical extraction from the cell biomass and quantitative determination of anthocyanin fractions using spectrophotometry. Key words anthocyanin, suspension cell culture, cell biomass, Vitis vinifera L., spectrophotometry Anthocyanins are natural pigments that demonstrate homeostatic, hypotensive, cardioprotective and anti-tumor properties [8,12]. 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 [4,5,13]. The research initiated for the six grapevine (Burgund Mare, Cabernet Sauvignon, Merlot, Oporto, Negru Tinctorial and Pinot Noir) varieties consists in obtaining of anthocyanins [14,15] from cell biomass [1,2,9,10] via suspension cell cultures [11]. Biological Material and Method In case of suspension cell cultures in the laboratory bioreactor, the culture was initiated from an inoculum s culture of 100 ml which has been transferred after 3 days of cultivation in the incubator and under shaking into the bioreactor. The bioreactor temperature was controlled while culture shaking was performed by air ebullition [6]. 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. 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 The UV-VIZ spectra were traced for the wave length domain 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 Results and Discussions Using suspension cell culture in the laboratory bioreactor, we have observed that after 15 days of culture, the amount of synthesized anthocyanins recorded values between 0,892 mg/g for Cabernet Sauvignon variety and 9,656 mg/g for Negru Tinctorial variety, with a magnitude of variation - 8,764 mg/g (Table 1). The effect of cell cultures in suspension in laboratory bioreactor on anthocyanin synthesis Cultivation duration (days) Average Negru Tintorial 9,656+0,081 10,816+0,229 10,236+0,281 Pinot Noir 2,157+0,057 2,363+0,101 2,260+0,069 Burgund Mare 5,335+0,035 5,516+0,109 5,425+0,065 Oporto 4,482+0,190 4,665+0,225 4,573+0,138 Merlot 2,454+0,129 2,590+0,068 2,521+0,072 Cabernet Sauvignon 0,892+0,032 0,868+0,121 0,879+0,056 Media 4,162+0,700 4,469+0,780 Valoarea relativă (%) 100,00 107,38 Diferenţa/Semnificaţia - 0,307 DL 5% =0,42 DL 1% =0,97 DL 0,1% =3,08 Table 1 443

2 After 30 days of cultivation in the laboratory bioreactor condition, synthesized anthocyanins had registered values between 0,868 10,816 mg/g, values recorded for those varieties with 9,948 mg/g. variation amplitude. The biosynthetic potential of cells cultivated in the laboratory bioreactor varied between 0,87 mg anthocyanins/g biomass for Cabernet Sauvignon and 10,23 mg anthocyanin/g biomass for Negru Tinctorial variety (Table 2). Negru Tinctorial variety recorded the higher values which were very significantly for the anthocyanins amount synthesized in cell cultures of all varieties, followed by Burgund Mare (5,42 mg/g), Oporto (4.57 mg/g), Merlot (2,52 mg/g) and Pinot Noir (2,26 mg/g) varieties. The differences recorded between the varieties were in most cases very significant. Table 2 The comparison of variety mean regarding the synthesized anthocyanins using suspension cell culture in bioreactor Pinot N Negru T 2,26 10,24 22,08-7, Burgund M Negru T 5,43 10,24 53,00-4, Oporto Negru T 4,57 10,24 44,68-5, Merlot Negru T 2,52 10,24 24,63-7, Cabernet S Negru T 0,88 10,24 8,60-9, Burgund M Pinot N 5,43 2,26 240,06 3,17*** Oporto Pinot N 4,57 2,26 202,35 2,31*** Merlot Pinot N 2,52 2,26 111,57 0,26* Cabernet S Pinot N 0,88 2,26 38,93-1, Oporto Burgund M 4,57 5,43 84,29-0, Merlot Burgund M 2,52 5,43 46,48-2, Cabernet S Burgund M 0,88 5,43 16,22-4, Merlot Oporto 2,52 4,57 55,14-2, Cabernet S Oporto 0,88 4,57 19,24-3, Cabernet S Merlot 0,88 2,52 34,90-1, DL 5% =0,26 DL 1% =0,35 DL 0,1% =0,4 a) The comparison of studied grape varieties regarding the synthesized anthocyanins using suspension cell culture in bioreactor after 15 days Table 3 Pinot N Negru T 2,16 9,66 22,34-7, Burgund M Negru T 5,33 9,66 55,25-4, Oporto Negru T 4,48 9,66 46,41-5, Merlot Negru T 2,45 9,66 25,41-7, Cabernet S Negru T 0,89 9,66 9,23-8, Burgund M Pinot N 5,33 2,16 247,32 3,18*** Oporto Pinot N 4,48 2,16 207,77 2,32*** Merlot Pinot N 2,45 2,16 113,75 0,30 Cabernet S Pinot N 0,89 2,16 41,33-1, Oporto Burgund M 4,48 5,33 84,01-0, Merlot Burgund M 2,45 5,33 45,99-2, Cabernet S Burgund M 0,89 5,33 16,71-4, Merlot Oporto 2,45 4,48 54,75-2, Cabernet S Oporto 0,89 4,48 19,89-3, Cabernet S Merlot 0,89 2,45 36,33-1,

3 b) The comparison of studied grape varieties regarding the synthesized anthocyanins using suspension cell culture in bioreactor after 30 days Pinot N Negru T 2,36 10,82 21,85-8, Burgund M Negru T 5,52 10,82 51,00-5, Oporto Negru T 4,67 10,82 43,13-6, Merlot Negru T 2,59 10,82 23,94-8, Cabernet S Negru T 0,87 10,82 8,03-9, Burgund M Pinot N 5,52 2,36 233,42 3,15*** Oporto Pinot N 4,67 2,36 197,40 2,30*** Merlot Pinot N 2,59 2,36 109,58 0,23 Cabernet S Pinot N 0,87 2,36 36,75-1, Oporto Burgund M 4,67 5,52 84,57-0, Merlot Burgund M 2,59 5,52 46,95-2, Cabernet S Burgund M 0,87 5,52 15,74-4, Merlot Oporto 2,59 4,67 55,51-2, Cabernet S Oporto 0,87 4,67 18,62-3, Cabernet S Merlot 0,87 2,59 33,53-1, DL 5% =0,36 DL 1% =0,49 DL 0,1% =0,67 The anthocyanin synthesis in cell cultures in bioreactor showed that both, the 15 days (Table 3a) and at 30 days (Table 3b) of culture, the hierarchy of grapevine varieties for anthocyanin synthesis remains unchanged, with very significant differences between varieties. According to data from table 4, only for the Negru Tinctorial variety we observed distinct significant differences for synthesized anthocyanins determinations made after 15 and 30 days. The comparison of variety mean regarding the anthocyanin synthesized using suspension cell culture in bioreactor Table 4 Cultivation duration Negru Tintorial ,82 9,66 112,01 1,16** Pinot Noir ,36 2,16 109,56 0,21 Burgund Mare ,52 5,33 103,40 0,18 Oporto ,67 4,48 104,09 0,18 Merlot ,59 2,45 105,54 0,14 Cabernet Sauvignon ,87 0,89 97,41-0,02 DL 5% =0,50 DL 1% =0,89 DL 0,1% =2,13 Conclusions 1. The biosynthetic potential of cells cultivated in the laboratory bioreactor varied between 0,87 mg anthocyanins/g biomass for Cabernet Sauvignon and 10,23 mg anthocyanin/g biomass for Negru Tinctorial variety. 2. The largest biosynthetic potential has been registered for Negru Tinctorial variety. 3. The mean values of the anthocyanin amount synthesized by the cells of this variety cultivated into the bioreactor were very significantly superior to the values registered for all studied grapevine varieties. 4. Considering the cell suspensions cultivated into the bioreactor, there were registered 5,42 mg/g, for Burgund Mare, 4,57 mg/g, for Oporto, 2,52 mg/g, for Merlot and 2,26 mg/g, for Pinot Noir varieties. 5. The differences registered between variety means were in most cases very significant. 6. Considering also the cell culture in the bioreactor, the growth rate and the biosynthetic potential were strongly 445

4 References influenced by genotype. The highest values for growth rate were registered for Negru Tinctorial and Burgund Mare varieties, while the highest cell production was attained in suspensions for Negru Tinctorial variety. 1. Gamborg, O.L., Murashige, T., Thorpe, T.A., Vasil, I.K., 1976, Plant tissue culture media, In Vitro, 7, p 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 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 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 Lazăr, A., Nedelea G., 2008, In vitro response of Vitis vinifera L. cell suspension cultures under the condition of their cultivation in the laboratory bioreactor, Cercetări ştiinţifice seria a XII-a, Horticultură Inginerie genetică, Editura Agroprint, Timişoara, p Lazăr, A., 2008, Researches concerning the obtaining of some secondary metabolites using in vitro cultures in Vitis vinifera L., Teza de doctorat, Timişoara, p 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 Markakis, P., 1982, Antocyanins as Food Colours. New York: Academic Press, p Murashige, T., Skoog, F., 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant., 15, p Oluf, L.C., Jerry, P.S., 1981, Nutrition, Media and Characteristics of Plant Cell and Tissue cultures, Plant Tissue Culture, Ed. Academic Press, p Payne, G. F., Bringi, V., Prince, C., Shuler, M. L., 1991, Suspension culture -Plant Cell and Tissue Culture in Liquid Systems, Hanser, p Rao, S.R., Ravishankar, G.A., 2002, Plant cell cultures: Chemical factories of secondary metabolites, Biotech. Advances 20, p Tărăbăşanu-Mihailă, C.N., Gorduza, V.M., Radu, F., Măzgăreanu, M., 1997, Coloranţi organici de interes alimentar, cosmetic şi farmaceutic. Bucureşti: Uni-Press, p 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), Yamakawa T., Kato S., Ishida K., Kodama T., Minoda Y., 1983, Production of anthocyanins by Vitis cell in Suspension Culture, Agr. Biol. Chem., 47(10),

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Experimental results concerning the effect of photoperiod and callus culture duration on anthocyanin amount

Experimental results concerning the effect of photoperiod and callus culture duration on anthocyanin amount Lazăr A. 1 *, Petolescu Cerasela 1, Popescu Sorina 1 1 USAMVB Timişoara, Faculty of Horticulture