Transgenic hairy root in vitro culture system of apple as a tool for allelopathic studies

Abstract

Since the investigation of plant allelopathic interactions in the rhizosphere is difficult to perform because of its high complexity, microbial and environmental factors interactions, the generation of autonomous root culture could be appropriate system for the studies of allelopathic potential of apple (Malus x domestica Borkh.). Thus, we have focused our efforts on the generation of transformed hairy-roots in apple cv. Golden Delicious using the 15834 strain of Agrobacterium rhizogenes. The agrobacteria inoculation by dipping the basal cut off end of the stem of in vitro grown apple shoots into bacterial suspension proved to be the best option for transformation, providing efficiency of 39.3%. Molecular characterization of hairy roots by RT-PCR analyses confirmed their transgenic nature. In vitro conditions for apple hairy root growth and maintenance were optimized using different mineral solutions and auxins. The liquid half-strength MS medium with the addition of IBA at 0.025 mg L-1 was chosen for the initial stimulation of growth. For their prolonged cultivation and maintenance, the alternation of IBA-containing and IBA-free media is recommendable. Fast-growing lines continuously maintained in IBA-free medium were used for further research. Potential allelochemicals including phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (phloridzin, rutin, quercitrin, isoquercitrin, kaempferol- 3-glucoside) and flavonoid aglycons (floretin, quercetin and naringenin) have been detected by UHPLC/DAD/(+/-)HESI-MS/MS analyses in the growth medium, in which these transformed roots were maintained for 4 weeks, as well as in the root tissue itself. The obtained hairy-root cultures offer a complementary system for studying and validating the allelopathic potential of apple roots.