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. e-mail: 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 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 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) 15 30 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
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 22,08-7,98 000 Burgund M Negru T 5,43 10,24 53,00-4,81 000 Oporto Negru T 4,57 10,24 44,68-5,66 000 Merlot Negru T 2,52 10,24 24,63-7,71 000 Cabernet S Negru T 0,88 10,24 8,60-9,36 000 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 38,93-1,38 000 Oporto Burgund M 4,57 5,43 84,29-0,85 000 Merlot Burgund M 2,52 5,43 46,48-2,90 000 Cabernet S Burgund M 0,88 5,43 16,22-4,55 000 Merlot Oporto 2,52 4,57 55,14-2,05 000 Cabernet S Oporto 0,88 4,57 19,24-3,69 000 Cabernet S Merlot 0,88 2,52 34,90-1,64 000 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 22,34-7,50 000 Burgund M Negru T 5,33 9,66 55,25-4,32 000 Oporto Negru T 4,48 9,66 46,41-5,17 000 Merlot Negru T 2,45 9,66 25,41-7,20 000 Cabernet S Negru T 0,89 9,66 9,23-8,76 000 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 41,33-1,27 000 Oporto Burgund M 4,48 5,33 84,01-0,85 000 Merlot Burgund M 2,45 5,33 45,99-2,88 000 Cabernet S Burgund M 0,89 5,33 16,71-4,44 000 Merlot Oporto 2,45 4,48 54,75-2,03 000 Cabernet S Oporto 0,89 4,48 19,89-3,59 000 Cabernet S Merlot 0,89 2,45 36,33-1,56 000 444
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 21,85-8,45 000 Burgund M Negru T 5,52 10,82 51,00-5,30 000 Oporto Negru T 4,67 10,82 43,13-6,15 000 Merlot Negru T 2,59 10,82 23,94-8,23 000 Cabernet S Negru T 0,87 10,82 8,03-9,95 000 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 36,75-1,49 000 Oporto Burgund M 4,67 5,52 84,57-0,85 000 Merlot Burgund M 2,59 5,52 46,95-2,93 000 Cabernet S Burgund M 0,87 5,52 15,74-4,65 000 Merlot Oporto 2,59 4,67 55,51-2,08 000 Cabernet S Oporto 0,87 4,67 18,62-3,80 000 Cabernet S Merlot 0,87 2,59 33,53-1,72 000 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 30-15 10,82 9,66 112,01 1,16** Pinot Noir 30-15 2,36 2,16 109,56 0,21 Burgund Mare 30-15 5,52 5,33 103,40 0,18 Oporto 30-15 4,67 4,48 104,09 0,18 Merlot 30-15 2,59 2,45 105,54 0,14 Cabernet Sauvignon 30-15 0,87 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
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