Alelopatski potencijal sekundarnih metabolita transformisanih korenova jabuke - efekat floretina i florizina u kulturi in vitro

Abstract

Ograniĉenja u izuĉavanju potencijala i mehanizama alelopatskog delovanja glavnih sekundarnih metabolita korena jabuke, floretina i florizina, usled kompleksnosti interakcija u rizosferi, mogu biti prevaziĊena upotrebom kulture transformisanih korenova in vitro. Odabranim naĉinom inokulacije regenerisanih izdanaka jabuke sa Agrobacterium rhizogenes 15834, efikasno su indukovani transformisani korenovi kod sorti Melrouz, Zlatni Delińes, Ĉadel i Gloster. Smenom hranljivih podloga sa i bez auksina, po prvi put je uspostavljena permanentna in vitro kultura izolovanih transformisanih korenova jabuke. UHPLC/(+/-)HESI–MS/MS analizom je utvrĊeno da genetiĉka transformacija nije dovela do promena u sadrņaju glavnih sekundarnih metabolita korena jabuke. U eksudatu transformisanih korenova, sadrņanom u teĉnoj podlozi u kojoj su korenovi gajeni, kvantifikovani su iskljuĉivo florizin, floretin, hlorogena i kafeinska kiselina. Tretman ovim eksudatom je prouzrokovao znaĉajnu inhibiciju rastenja i razvića izdanaka i korenova klijanaca Arabidopsis thaliana. Sliĉan inhibitorni efekat ispoljili su ĉist florizin u koncentraciji od 500 μM, kao i floretin u svim testiranim koncentracijama (125-500 μM), izazivajući krupne promene u morfologiji tretiranih klijanaca, posebno korena. Velike sliĉnosti su zapaņene u obrascu promena ekspresije gena ukljuĉenih u polarni transport auksina, kao i biosintezu auksina i giberelina u korenovima klijanaca arabidopsisa tretiranih eksudatom transformisanih korenova jabuke, sa onim detektovanim kod klijanaca tretiranih 500 μM floretinom. To ukazuje na mogućnost da je floretin u eksudatu korena jabuke metabolit koji je odgovoran za alelopatske efekte, a koji bi se mogli zasnivati na promenama nivoa auksina i giberelina, kao i na interakciji ove dve grupe hormona.

The investigation of allelopathic potential and mode of action of the main apple root secondary metabolites, phloridzin and phloretin is difficult to perform due to high complexity of rhizosphere interactions. Therefore, the generation of in vitro hairy root cultures could be an alternative system for allelopathic studies of apple. The efficient induction of hairy roots in apple cvs. Melrose, Golden Delicious, Ĉadel and Gloster was obtained using the appropriate mode of inoculation of regenerated shoots by Agrobacterium rhizogenes 15834. Permanent in vitro culture of apple autonomous hairy roots was established for the first time by cultivation on alternating growth media with and without auxins. UHPLC(+/-)HESI-MS/MS analysis confirmed that genetic transformation did not lead to changes in the content of the main secondary metabolites of apple roots. Dihydrochalcones phloridzin and phloretin, chlorogenic and caffeic acids were detected as only putative allelochemicals exuded into the growth medium in which hairy roots were grown. Apple hairy root exudates significantly affected development and growth of Arabidopsis thaliana seedlings. Pure phloridzin at 500 μM and phloretin in all tested concentrations (125-500 μM) had similar effects as apple hairy root exudates inducing significant alterations in morphology of seedlings, expecially roots. High similarities in the expression patterns of genes involved in polar auxin transport and auxin and gibberellin biosynthetic pathways in roots of arabidopsis seedlings treated with either apple hairy root exudates or 500 μM phloretin were observed. This indicates that phloretin in hairy root exudates could be at least partly responsible for the apple root allelopathic properties, that were most likely based on auxin and gibberellin cross-talk and their altered homeostasis in arabidopsis roots.