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Genetic transformation of gentian Gentiana tibetica (King) leaf explants with Agrobacterium tumefaciens strain C58C1

Research paper by Anna I. Wójcik, Jan J. Rybczyński

Indexed on: 28 Dec '16Published on: 22 Dec '16Published in: Acta Physiologiae Plantarum



Abstract

Abstract Experiments were carried out on transformation of leaf explants derived from the axenic culture of Gentiana tibetica (King) using a co-culture of Agrobacterium tumefaciens. A. tumefaciens octopine strain C58C1 carried neomycine phosphotransferase (nptII) and β-glucuronidase (uidA) genes. The influence of plasmid helper pCH32 on transformation efficiency was stressed. After co-cultivation, explant was cultured on three different regeneration media (RM1-3) at the presence of the timentin and callus formation and plants regeneration occurred. The transgenic character of the selected tissue and transformants T0 in the presence of kanamycin has been confirmed by the histo-chemical analysis of reporter enzyme activity (ß-glucuronidase), polymerase chain reaction, and southern hybridization detecting uidA and nptII genes. l-glutamine used during inoculation period had a significant influence on the transformation efficiency in relation to its concentration and time of treatment.AbstractExperiments were carried out on transformation of leaf explants derived from the axenic culture of Gentiana tibetica (King) using a co-culture of Agrobacterium tumefaciens. A. tumefaciens octopine strain C58C1 carried neomycine phosphotransferase (nptII) and β-glucuronidase (uidA) genes. The influence of plasmid helper pCH32 on transformation efficiency was stressed. After co-cultivation, explant was cultured on three different regeneration media (RM1-3) at the presence of the timentin and callus formation and plants regeneration occurred. The transgenic character of the selected tissue and transformants T0 in the presence of kanamycin has been confirmed by the histo-chemical analysis of reporter enzyme activity (ß-glucuronidase), polymerase chain reaction, and southern hybridization detecting uidA and nptII genes. l-glutamine used during inoculation period had a significant influence on the transformation efficiency in relation to its concentration and time of treatment.Gentiana tibeticaAgrobacterium tumefaciens. A. tumefaciensnptIIuidAuidAnptIIl