Hairy root culture as a valuable tool for allelopathic studies in apple

Abstract

Allelopathic plants exploit their chemical ‘weapons’ to prevail over the competition, suppress neighboring plants and consequentlyuse the available resources more efficiently. However, the investigation of plant allelopathic interactions in rhizosphere is difficultto perform because of its high complexity due to interactions of biotic and abiotic factors. Thus, autonomous, aseptic root culturesof apple (Malus × domestica Borkh.) could facilitate allelopathic studies. We report on the successful genetic transformation ofapple cultivars Melrose, Golden Delicious, Cadel and Gloster using ˇ Agrobacterium rhizogenes (Riker et al. 1930) Conn 1942 strain15834 and for the first time the establishment of apple autonomous and permanent in vitro hairy root cultures that could be usedas a new tool for apple allelopathic assays. Molecular characterization of transgenic hairy root lines was conducted to elucidatethe possible relationship between expression of T-DNA genes and root growth characteristics that include branching. Similar content of phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (rutin, quercitrin, isoquercitrin,kaempferol-3-glucoside) and flavonoid aglycones (quercetin and naringenin), and dihydrochalcone phloridzin, was detected inuntransformed and transgenic apple root tissue by ultra high-performance liquid chromatography with mass spectrometry(UHPLC/(+/–)HESI–MS/MS) analyses, confirming that genetic transformation did not disturb secondary metabolite production inapple. Chlorogenic and caffeic acids and dihydrochalcones phloridzin and phloretin were detected as putative allelochemicalsexuded into the growth medium in which transgenic roots were maintained for 4 weeks. Apple hairy root exudates significantlyaffected shoot and root development and growth of test plant Arabidopsis thaliana (L.) Heynh. seedlings after 5 or 10 days oftreatment. Additionally, core cell-cycle genes CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 were down regulated in Arabidopsisshoots suggesting, in part, their role in inhibition of shoot growth. The present work highlighted an autonomous and permanentin vitro hairy root culture system as a valuable tool for studying allelopathic potential of apple, offering new perspective for allelopathy background elucidation in this important fruit species.