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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011genotype (1) / 10 000 2.00 ± 0.0000 4.00 ± 0.0000 7.00 ± 1.4142ArMV +GFkV Control 1.00 ± 0.0000 6.00 ± 0.0000 a 6.50 ± 0.7071Unidentified 1000 1.00 ± 0.0000 2.00 ± 1.4142 3.65 ± 0.4950genotype (2) / 10 000 1.50 ± 0.7071 2.50 ± 0.7071 3.25 ± 0.3536GFLV+ Control 1.00 ± 0.0000 2.00 ± 0.0000 3.50 ± 0.7071GLRaV-1The values are means± sd on three repetitions, the small letters representthe significance of the differences between variants as compared to the control at P < 0.05Plant regeneration, in general, depends upon several factors, including: genotype,physiological state of the explant, culture medium, the cultivation conditions and theinteractions betwen these factors (D. SVETLEVA & al. [18]). The electric pulses are olsoreported as stimulant of in vitro plant differentiation (A. GOLDSWORTHY [19]). It wasdemonstrated that regeneration of potato plant tissues could be improved by exposingexplants to mild electric current (H.F. LOZOYA-SALDAÑA & al. [10]).Alternating electric currents (50 Hz, 0.1–50 µA) applied for 30 days on Nicotianatabacum calli induced a significant stimulation of morphogenetic processes. The numberof shoots increased by up to 300% for the samples stimulated with 50 µA but nosignificant changes were noted in total mass, DNA and protein content in the stimulatedsamples compared to control. It was suggested that in the presence of the external electriccurrent, the callus cells become more sensitive to chemical signals (hormones and/or ions)in the culture medium (G. COGĂLNICEANU & al. [20]).After three subculture on multiplication media, differentiated and elongatedmicroshoots have been transfered on rooting medium. In general, the rhyzogenesisassessed by the number of rooted microshoots took place without large differences on theexperimental variants. In the case of Fetească neagră cv. initially infected with GLRaV-1, asignificant positive difference have been registered, in that the treatment with 10 000 kHzdetermined the increasing of the number of rooted shoots (121± 0.8112) comparativelywith the control (69± 8.4853) (Table 3).Significant increases in rooting rate and root growth parameter indicated thatdirect electric current applications may have the potential to improve propagation ofgrapevine rootstocks that are difficult to root by cuttings (C. Köse [21]). On the other hand,higher intensities of electric current adversely affected the survival of bean explants invitro and, thus, the plant regeneration (M.H. HORMOZI-NEJAD [22].Table 3. In vitro rooting assessed by the number of rooted shoots on the experimental variantsGenotype/VirusFrequency(kHz)Rooted shoots(N o )46Fetească albă /1000 11.00 ± 5.6569GFLV10 000 19.00 ± 4.2426Control 15.50 ± 0.7071Fetească neagră /1000 71.50 ± 28.9914GLRaV-110 000 121.00 ± 0.8112 cControl 69.00 ± 8.4853 aFrâncuşă / 1000 20.00 ± 21.2100
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011GLRaV-110 000 21.00 ± 19.8000Control 18.00 ± 2.8300Cabernet Sauvignon /1000 13.00 ± 0.0000GLRaV-310 000 17.00 ± 1.4100Grasă de Cotnari /GFkVUnidentified genotype (1) /ArMV + GFkVUnidentified genotype (2) /GFLV + GLRaV-1Control 18.50 ± 2.12001000 36.00 ± 8.490010 000 56.00 ± 29.7000Control 42.00 ± 2.83001000 6.00 ± 1.414210 000 7.00 ± 1.4142Control 17.00 ± 1.41421000 19.00 ± 1.410010 000 20.00 ± 4.2400Control 19.50 ± 0.7100The values are means± sd on three repetitions, the small letters represent the significanceof the differences between variants as compared to the control at P < 0.05Virus elimination assessmentThe efficacy of virus elimination process assessed by ELISA testing ofregenerated acclimated grapevine plants was presented in Table 4.The analysis showed some encouraging results for elongated viruses GLRaV-1and GLRaV-3 in single infections and no virus eradication in mixed infections had beenobtained. The electrotherapy of GLRaV-1- infected material produced maximum 12.5%virus-free plants at 1000 kHz for and 4.5% GLRaV-3-free vines at 10 000 kHz, for 10 minof exposure. Grasă de Cotnari variety was 100% GFkV-free in the case of 10 000 kHzvariant. The results regarding GLRaV-1 or GFkV elimination were not reproducible in thecase of mixed infection containing one of them. In the case of 1000 kHz variants, anyacclimated vine has been obtained for cv. Frâncu ă and unidentified genotype (2). Noșsatisfactory results have been achieved for isometric viruses eradication (GFLV, GFkV)either in simple or mixed infections. ArMV has been eradicated in proportion of 85.7 –88.9% but the vines belonging to unidentified genotype (1) remained GFkV infected.Table 4. Assessment of efficiency of grapevine virus elimination by electrotherapywith direct electric curentGrapevine cultivar / FrequencyPlants (N o )Sanitationvirus(kHz) ELISA Virus-free ratetested (N o ) (N o )(%)Fetească albă /1000 20 0 0GFLV 10 000 17 0 0Fetească neagră /1000 66 0 0GLRaV-1 10 000 146 0 01000 8 1 12.5ș47
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