Adventitious shoot regeneration from leaves of peach cultivars growing in vitro: the effect of cytokinins

In the present study the effect of different cytokinins on adventitious shoot regeneration in two peach cultivars, ‘Rich Lady’ and ‘Independence’, was evaluated. In vitro shoots were grown for one subculture on a multiplication medium containing 5, 10 or 15 mg L^-1 meta-Topolin (mT), an aromatic natural cytokinin, or benzyladenine (BA), then apical leaves were excised and transferred on a culture medium supplied with α-naphthaleneacetic acid (NAA) and BA, thidiazuron (TDZ), mT or zeatin (ZEA) to induce adventitious regeneration. Leaves were cultured 21 days in darkness on these media and then transferred to the same auxin-free media under light conditions. During the dark incubation period, leaves developed a callus at the petiolar base. Adventitious shoots appeared on this callus in leaves excised from shoots previously grown both on mT and BA. In ‘Rich Lady’, the highest regeneration response (32%) was obtained either with the pre-treatment with 10 mg L^-1 BA and applying BA during the regeneration phase and with 5 mg L^-1 mT and applying mT in the regeneration phase. The highest regeneration responses in ‘Independence’ were till 19% using 10 mg L^-1 mT as pre-treatment and ZEA and mT in the regeneration medium, or 15 mg L^-1 mT as pre-treatment and TDZ during regeneration. These results appear promising for the application of cisgenesis and genome editing by CRISPR/Cas9 approach, aimed to genetic improvement of productivity and resistance of peach cultivars in the frame of the project “BIOTECH”, sub-project “Biotechnology for sustainable fruit crops” (BIOSOSFRU), recently financed by the Italian Agriculture Ministry.