For breeding purposes, the number of neo-tetraploids of apple cultivars have been derived by in vitro technique. The first attempts at rooting and acclimatization of tetraploid shoots failed. The aim of the study was to develop an effective method for rooting microcuttings of apple neo-tetraploids. In the first stage of the study, in vitro rooting method was optimized for the shoots of diploid donor cultivars. Shoots were rooted on Murashige and Skoog [1962] (MS) medium with a reduced content of nitrogen, in the presence of auxins alone or in combination (indole-3-butyric acid – IBA, 1-naphthaleneacetic acid – NAA and indole- -3-acetic acid – IAA) with addition of putrescine, arginine or ornithine. The compounds were applied continuously for 25 days (one-step rooting system) or for seven days with subsequent transplanting shoots onto a medium without these compounds (two-step rooting method). Tetraploid microcuttings of the cultivars ‘Free Redstar’, ‘Gala Must’, ‘Pinova’ and ‘Redchief’ were evaluated for rooting on the selected medium considered optimal for their diploid counterparts. The shoots of all diploid apple scion cultivars had low rooting capacity. IBA alone poorly stimulated root formation. Significant improvement of rooting to 60–80% was achieved through the application of auxins, 2.5 µM IBA or 1.3 µM NAA combined with 5 µM IAA and 50 µM putrescine in the two-step rooting system with darkness and increased temperature of 26°C during seven-day induction phase. The replacement of benzyladenine (BA) by meta-Topolin (m-T) in the last multiplication subculture influenced positively shoot acclimatization. Tetraploids had comparable or slightly lower rooting and acclimatization ability compared to their diploid counterparts.

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