New Researches in Biotechnology - Facultatea de Biotehnologii ...
New Researches in Biotechnology - Facultatea de Biotehnologii ... New Researches in Biotechnology - Facultatea de Biotehnologii ...
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011plants, therefore the use virus-free vines for multiplication and new vineyard establishingis required (F. MANNINI [1]). In cases of high incidence of infectious diseases in breedingvineyards and impossibility to select authentic and disease-free material of a particularvariety or rootstock, several techniques are being applied for virus elimination (I.TSVETKOV & al. [ 2]).The meristem culture and thermotherapy are individually or in combination themost frequent methods of obtaining virus-free grapevines (G. GRAMMATIKAKI & A.AVGELIS [3]; B. KŘIŽAN & al. [4]) The application of heat treatment for viruseliminationin plants is time and energy consuming and needs special equipments(chambers for heat treatment). In the case of the meristem culture, the percent of viruselimination is influenced by the genotype, the type of the virus and especially of thedifficulty of meristem excision.Virus-elimination techniques such as: chemotherapy (A. PANATTONI & al.[5]), electrotherapy in continuous electric field (I.C. GUȚĂ & al. [6]), cryotherapy (SH.BAYATI & al. [7]), somatic embryogenesis (G. GAMBINO & al. [8]) and theircombinations are considered alternative method to thermotherapy. Each of the listedmethod involves one stage of in vitro culture.Is well known the use of electric current in the cleaning of viral diseases in plants;instead, a deep knowledge of this phenomenon theoretical basis is not yet available. Theexperiments demonstrated that the cleaning process nature is, essentially, the effect of theviral protein denaturation, by means of the heat to which they were exposed in the thermalbath that the vegetal tissue constitutes (J.E GONZÁLES & al. [9]).Electrotherapy has been applied successfully to eradicate potato virus X fromdifferent infected potato clones (H.F. LOZOYA-SALDAÑA & al. [10]); dasheen mosaicvirus in malanga (J. IGARZA CASTRO & al. [11]; banana streak virus in banana (P.R.HERNÁNDEZ & al. [12]).The electrotherapy for GFLV elimination was investigated, as a more practicalalternative technique comparatively to heat therapy of virus infected grapevine, but notsatisfactory results have been obtained. However, the decreasing of by enzyme-linkedimmunosorbent assay (ELISA) values in function of the exposure period have beenregistered (J.G. BURGER [13]).The electrotherapy followed by in vitro culture of shoot apices produced 33.33–66.66% GLRaV-1+3 –free plants, regardless explant position of exposed grapevine incontinuous electric field (I.C. GUȚĂ & al. [14]).The aim of the present research was to eliminate fanleaf virus (GFLV), arabismosaic virus (ArMV), leafroll associated virus 1 (GLRaV-1), leafroll associated virus 342
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011(GLRaV-3), and fleck virus (GFkV) in simple or mixed infections at a time, in seven Vitisvinifera L. varieties, by electrotherapy with direct electric current.2. MATERIALS AND METHODSSource of virus-infected material: The study regarding GFLV, ArMV, GLRaV-1,GLRaV-3, GFkV and their combinations (virus complex) elimination has been done onseven V. vinifera L. cultivars naturally infected (Table 1), maintained in the grapevinevirus infected collection of NRDIBH Ştefăneşti–Argeş. One year old infected plantsobtained from one bud cuttings, confirmed as virus-infected by ELISA testing, constitutedthe source of nodal fragments (microshoots).Table 1. Virus-infected grapevine cultivars used in the experimentGrapevine cultivarVirus/virus complexFetească albăGFLVFetească neagrăGLRaV-1GLRaV-1FrâncușăCabernet SauvignonGrasă de CotnariUnidentified genotype (1)Unidentified genotype (2)GLRaV-3GFkVArMV + GFkVGFLV + GLRaV-1Electrotherapy: In the case of electrotherapy with direct electric current, a sourcethat converts the alternating current from electricity network in alternating current withadjustable parameter is required. The experiment consists of subjecting a nodal fragment toan alternating current for a certain period, after that it is inoculated on culture medium,being the source of regeneration of new grapevine plants.The experimental parameter were established after previous attempts (directelectric current of 1; 100; 1000 and 10 000 kHz was applied for 5; 10 and 20 min at thecut ends of one budded herbaceous cuttings), regarding the viability of microshootsexposed to the action of electric current (unpublished data). The current frequency (1000kHz; 10 000 kHz) and exposure time (10 min) for this experiment were established. Thus,the experience is bifactorial of subdivided parcels type, 2x7 experimental variants; for eachvariant were exposed three microshoots as repetitions.The control for each variant was represented by virus infected microshoots in vitrocultured unexposed to direct electric curent (untreated). The position of the nodal fragmentson the shoots were identically for all experimental variants, namely the second internodebelow the apex.In vitro culture: Nodal fragments subjected to electrical current treatment andvirus- infected untreated nodat fragments (control) have been inoculated on M&S (1962)basic medium (T. MURASHIGE & F. SKOOG [15]) containing 2 µmol L -1benzylaminopurine (BAP) and 5 µmol L -1 indolilacetic acid (AIA). After three subcultures43
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Proceed<strong>in</strong>g of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011(GLRaV-3), and fleck virus (GFkV) <strong>in</strong> simple or mixed <strong>in</strong>fections at a time, <strong>in</strong> seven Vitisv<strong>in</strong>ifera L. varieties, by electrotherapy with direct electric current.2. MATERIALS AND METHODSSource of virus-<strong>in</strong>fected material: The study regard<strong>in</strong>g GFLV, ArMV, GLRaV-1,GLRaV-3, GFkV and their comb<strong>in</strong>ations (virus complex) elim<strong>in</strong>ation has been done onseven V. v<strong>in</strong>ifera L. cultivars naturally <strong>in</strong>fected (Table 1), ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> the grapev<strong>in</strong>evirus <strong>in</strong>fected collection of NRDIBH Ştefăneşti–Argeş. One year old <strong>in</strong>fected plantsobta<strong>in</strong>ed from one bud cutt<strong>in</strong>gs, confirmed as virus-<strong>in</strong>fected by ELISA test<strong>in</strong>g, constitutedthe source of nodal fragments (microshoots).Table 1. Virus-<strong>in</strong>fected grapev<strong>in</strong>e cultivars used <strong>in</strong> the experimentGrapev<strong>in</strong>e cultivarVirus/virus complexFetească albăGFLVFetească neagrăGLRaV-1GLRaV-1FrâncușăCabernet SauvignonGrasă <strong>de</strong> CotnariUni<strong>de</strong>ntified genotype (1)Uni<strong>de</strong>ntified genotype (2)GLRaV-3GFkVArMV + GFkVGFLV + GLRaV-1Electrotherapy: In the case of electrotherapy with direct electric current, a sourcethat converts the alternat<strong>in</strong>g current from electricity network <strong>in</strong> alternat<strong>in</strong>g current withadjustable parameter is required. The experiment consists of subject<strong>in</strong>g a nodal fragment toan alternat<strong>in</strong>g current for a certa<strong>in</strong> period, after that it is <strong>in</strong>oculated on culture medium,be<strong>in</strong>g the source of regeneration of new grapev<strong>in</strong>e plants.The experimental parameter were established after previous attempts (directelectric current of 1; 100; 1000 and 10 000 kHz was applied for 5; 10 and 20 m<strong>in</strong> at thecut ends of one bud<strong>de</strong>d herbaceous cutt<strong>in</strong>gs), regard<strong>in</strong>g the viability of microshootsexposed to the action of electric current (unpublished data). The current frequency (1000kHz; 10 000 kHz) and exposure time (10 m<strong>in</strong>) for this experiment were established. Thus,the experience is bifactorial of subdivi<strong>de</strong>d parcels type, 2x7 experimental variants; for eachvariant were exposed three microshoots as repetitions.The control for each variant was represented by virus <strong>in</strong>fected microshoots <strong>in</strong> vitrocultured unexposed to direct electric curent (untreated). The position of the nodal fragmentson the shoots were i<strong>de</strong>ntically for all experimental variants, namely the second <strong>in</strong>terno<strong>de</strong>below the apex.In vitro culture: Nodal fragments subjected to electrical current treatment andvirus- <strong>in</strong>fected untreated nodat fragments (control) have been <strong>in</strong>oculated on M&S (1962)basic medium (T. MURASHIGE & F. SKOOG [15]) conta<strong>in</strong><strong>in</strong>g 2 µmol L -1benzylam<strong>in</strong>opur<strong>in</strong>e (BAP) and 5 µmol L -1 <strong>in</strong>dolilacetic acid (AIA). After three subcultures43
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