Protocol for rapid in vitro shoot multiplication of ancient apple cultivars and landraces, grown in Czech Republic

Abstract

The apple fruit is the most economically important fruit species in the temperate climatic zone of central Europe. In 2012, a project was initiated to test the resistance of apple landraces and older cultivars to fire blight. The aim of this study was to develop a protocol for rapid in vitro shoot multiplication of selected apple cultivars. Successful in vitro propagation, which can be carried out all year round, could provide an alternative method to produce stock material of particular cultivars for experiments with artificial inoculation in safe and controllable laboratory conditions. Selected genotypes were successfully established in vitro using 0.15% mercuric chloride as disinfectant solution. To determine favourable conditions for shoot initiation and development, six proliferation MS media containing 1, 2 and 4 mg L(‑1) BAP (6-benzylaminopurine), 0.5 and 1 mg L(‑1) TDZ (thidiazuron) or 10 mg L(‑1) 2iP (6-(γ,γ-dimethylallylamino) purine) were tested. All apple genotypes in this study responded favourably to micropropagation in MS medium. The multiplication rate varied depending on the cultivar and concentration of cytokinins between 1.1 and 7.1. The highest proliferation rate was obtained for apple cultivar ‘Hvezdnata reneta’ which produced 7.1 in vitro shoots (longer than 10 mm) on MS medium containing 1 mg L(‑1) TDZ. On the contrary, the lowest proliferation rate 1.1 in our experiments was obtained for cultivar ‘Kminova reneta’ on MS medium with 10 mg L(‑1) 2iP. Our results confirmed the preliminary findings which indicated that TDZ was an important plant growth regulator for proliferation and growth in apple micropropagation. However, some abnormalities such as swollen shoots shorter than 10 mm with narrow leaves were observed during shoot multiplication on MS media with TDZ. The cytokinin BAP gave satisfactory results for multiplication in a concentration of 4 mg L(‑1). All cultivars had well-developed leaves on media containing BAP.