Advances in development of transgenic resistance to beet necrotic yellow vein virus (BNYVV) in sugar beet

Fragments of viral cDNA containing the coat protein gene of beet necrotic yellow vein virus were cloned in plant transformation vector pCAMBIA3301M with the bar gene as selectable marker. Vector pC3301MCPL carrying coat protein gene with leader sequence, and pC3301MCPS with coat protein gene, were used in Agrobacterium - mediated transformation of sugar beet. The transformation method used was based on the fact that sugar beet develops axillary shoots in in vitro conditions, when placed on media with citokinins. Since this ability is not genotype or ploidy dependant it is widely used for sugar beet vegetative multiplication. Sterile seedlings, with removed cotyledons and lower half of hypocotyl, were used as starting material. After transformation ex-plants were put on micropropagation medium with cephotaxime and phosphinotricyn (ppt), where axillary shoots started to develop. Since concentration of ppt was not selective enough, after two subcultivations it was increased twofold. Only one sample, transformed with pC3301MCPS preserved morphogenetic potential for micropropagatio, and it was tested for presence of COS fragment and bar gene bz PCR with soecific primers. Fragmenti virusne cDNK sa genom za protein omotaca virusa nekroticnog zutila nerava repe su klonirani u vektor za transformaciju biljaka pCAM-BIA3301M koji je sadrzao bar gen kao selektivni marker. Vektori pC3301MCPL, sa genom za protein omotaca virusa i njegovom lider sekvencom, i pC3301MCPS, sa genom za protein omotaca, su korisceni u tramsformaciji repe pomocu Agrobacterium-a. Metod transformacije se zasniva na sposobnosti repe da u uslovima in vitro razvije aksilarne pupoljke na podlozi sa citokininima. Posto ova sposobnost ne zavisi od genotipa ili od nivoa plodnosti, postala je standardni metod za vegetativno umnozavanje repe. Kao pocetni materijal su korisceni sterilni ponici kojima su odstranjeni kotiledoni i donja polovina hipokotila. Nakon transformacije eskplantati su postavljeni na selektivnu podlogu za mikropropagaciju sa cefotaksimom i fosfinotricinom (ppt) gde je doslo do razvoja bocnih pupoljaka. Po.sto koncentracija fosfinotricina nije bila dovoljno selektivna, ona je nakon dve subkultivacije dvostruko povecana. Samo je jedan uzorak, transformisan vektorom pC3301MCPS, nakon dve subkultivacije sacuvao mofrogenetski potencijal za mikropropagaciju, i bio testiran na prisustvo CPS fragmenta i bar gena PCR reakcijom sa specificnim prajmerima.