Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn.
Cytokinin oxidase/dehydrogenase (CKX) is the only known enzyme involved in cytokinin catabolism. Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were introduced separately into a binary cloning vector, immobilized into Agrobacterium tumefaciens strain GV3101, and introduced into root explants of centaury (Centaurium erythraea Rafn.). The integration of each transgene was confirmed by genomic PCR. Of the total transformed explants, 30 and 28.2 % of the transformants carried AtCKX1 and AtCKX2 transgenes, respectively. Of these transformants, 50 % exhibited expression of the AtCKX1 transgene, while 64 % of transformants exhibited expression of the AtCKX2 transgene. For all analysed AtCKX transgenic centaury lines, as well as for untransformed control plants, CKX activity was higher in roots than in shoots. Expression of AtCKX in most transgenic lines contributed to enhanced levels of CKX activity in root tissues; whereas, only a few lines demonstrated increased CKX activity in shoot tissues compared to those of control plants. Moreover, overexpression of AtCKX resulted in reduced morphogenetic potential in transgenic plants, but did not significantly affect biomass production in comparison to untransformed control plants.
KeywordsCentaurium erythraea Rafn. Agrobacterium tumefaciens Genetic transformation qRT-PCR AtCKX genes CKX activity
Luria–Bertani broth medium
Murashige and Skoog medium
The bacterial strains A. tumefaciens GV3101/pBinHTX-AtCKX1 and GV3101/pBinHTX-AtCKX were kindly provided by dr Thomas Schmülling and dr Tomáš Werner (Freie Universität Berlin, Germany). The authors thank Marie Korecká for her invaluable technical support. This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant No. ON173015) and the Czech Science Foundation (P506/11/0774).
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