Neem Oil to Reduce Zeatin Use and Optimize the Rooting Phase in Olea europaea L. Micropropagation

Micropropagation is an in vitro propagation technique, established in the nursery field sector for numerous species, which offers several advantages compared to traditional agamic propagation techniques. In the case of the olive tree, however, despite the advances made through research, it is still little used, due to the recalcitrance to in vitro proliferation and/or rooting of many olive cultivars and the high cost of zeatin, the only cytokinin that makes it possible to achieve a satisfactory proliferation rate in this species. In this context, numerous attempts have been made to identify alternative cytokinin compounds able to improve the proliferation rate of olive tree explants and thus reduce the unitary production cost. In particular, there is a growing interest in the use of natural substances (called in some cases "complex mixtures"), which, when added to the in vitro cultivation substrates, seem to be able to improve proliferation rates. In the present study, neem oil was added to the propagation substrates (partially/totally replacing zeatin) and in the rooting phase for the olive cultivar Moraiolo. In particular, in the proliferation phase, the effect of neem oil (0.1 mL L-1) in substrates containing different zeatin concentrations (0, 1, 2, and 4 mg L-1) was evaluated. For the rooting phase, agarized substrate and soil were used with shoots derived from a standard proliferation substrate (4 mg L-1 zeatin) and from the substrate that gave the best results in the proliferation phase (2 mg L-1 zeatin and 0.1 mL L-1 neem oil). In the proliferation phase, the addition of neem oil in the substrates with low zeatin concentration (1 and 2 mg L-1) induced an increase in the number of adventitious shoots and shoots length. On the contrary, the addition of neem oil in the rooting substrates did not positively influence the rooting phase, but positive results especially in terms of root number and length were observed in explants derived from a neem oil-enriched proliferation substrate compared to the control substrate. Therefore, the present study demonstrated for the first time the positive role of neem oil in the proliferation of olive in vitro with low zeatin concentrations.