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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011of 30 days each, when a multiplication process occurred, individualized and elongatedmicroshoots of 2-2.2 cm were cultured on rooting medium (E. VIȘOIU [16]). During theregenerative processes, the parameters such as multiplication rate (X) and number ofpercent of rooted microshoots on experimental variants were monitored.Virus elimination assessment: In order to assess the virus elimination efficiency,regenerated acclimated vines were analyzed by ELISA. The ELISA test was performedaccording to the method described by M.F. CLARK & A.N. ADAMS [17] withcommercial reagents produced by BIOREBA, Switzerland. Absorbance (OD 405nm ) wasrecorded by PR 3100 photometer. For a correct discrimination of negative and positiveresults, a cut-off value as three times the average values of negative control was calculated.Statistical assessment: Statistical analysis of the data have been made by SPSSfor Windows 10. In the tables, the values are means± standard deviations (sd). Thedifferences between variants as compared to the control were analyzed with one-wayANOVA-LSD taking P
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011a) the device of electrotherapy; b) direct stimulation of the microshoot;c) in vitro rooted grapevine plantlets; d) regenerated vines after electrotherapyThe evolution of the subcultures was assessed by the multiplication rate ascompared to the control for each infected grapevine genotype, depending on thecharacteristics of alternating current. In the first subculture, the virus infected genotypesbehaved no significant differences comparatively with untreated (controls); themultiplication rates range between 1.5000 ± 0.7071 and 3.0000 ± 1.4142. Starting from thesecond subculture, significant differences in the case of GLRaV-3–infected cv. CabernetSauvignon and ArMV+GFkV–infected unidentified genotype (1) have been occurred. Înthe first case, the difference was significantly negative at P< 0.05 for 10 000 kHzfrequency as compared with the control. Also, negative significaton for the double infectionArMV+GFkV at 1000 kHz has beeen found.After three subculture, the material has been stabilized; significant negativediffereces appeared to genotypes Fetească albă, Cabernet Sauvignon, Frâncu ă and Grasășde Cotnari. The data regarding the multiplication rates on three subculctures of infectedgrapevine genotype indicated that the electrotherapy with alternating current of variablefrequency influenced negatively the in vitro development of grapevine in some cases.This can have beneficial consequences for the purposes of possible viral inactivationmultiplication due to intracellular thermotherapy phenomenon produced by electriccurrent (Table 2).Table 2. The evolution of in vitro culture on experimental variants expressed by the multiplicationrate during three subcultures (I, II, III)Grapevine FrequencyMultiplication rate (X)genotype/ (kHz) I II IIIVirusFetească albă / 1000 1.50 ± 0.7071 3.00 ± 0.0000 8.50 ± 4.9497 bGFLV10 000 1.50 ± 0.7071 5.00 ± 1.4142 17.50 ± 4.9497Control 1.50 ± 0.7071 5.50 ± 2.1213 23.00 ± 4.2426 aFetească neagră 1000 1.50 ± 0.7071 5.50 ± 3.5355 16.50 ± 10.6066/ GLRaV-1 10 000 3.00 ± 1.4142 6.00 ± 1.4142 20.50 ± 0.7071Frâncuşă /GLRaV-1Control 2.50 ± 2.1213 3.50 ± 0.7071 15.50 ± 0.70711000 2.00 ± 1.4142 1.50 ± 0.7071 3.15 ± 0.212110 000 2.00 ± 0.0000 1.50 ± 0.7071 5.10 ± 0.8485 bControl 1.50 ± 0.7071 1.50 ± 0.7071 3.50 ± 0.7071 a1000 1.50 ± 0.7071 3.50 ± 0.7171 2.90 ± 0.4243 b10 000 3.00 ± 0.0000 b 2.10 ± 0.2121 b 1.60 ± 0.2828CabernetSauvignon /GLRaV-3 Control 1.50 ± 0.7071 a 4.70 ± 1.7678 a 1.45 ± 0.0070 aGrasă de Cotnari 1000 2.50 ± 0.7071 1.80 ± 0.2828 5.10 ± 0.1414 b/GFkV10 000 3.00 ± 0.0000 2.30 ± 0.4243 4.60 ± 0.5303Control 3.00 ± 0.0000 1.80 ± 0.2828 3.60 ± 0.4950 aUnidentified 1000 2.00 ± 0.0000 3.00 ± 0.0000 b 5.50 ± 0.707145
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