Effective regeneration in vitro is a necessary precondition for the implementation of different biotechnological approaches in plant breeding. Numerous studies have reported on regeneration from apple somatic tissues, and organogenesis has been proved to be influenced by several factors including mother shoots (genotype, size, type, and age of explant), in vitro conditions (dark period, light intensity, and quality), and others (wounding, orientation of leaf explants). However, one of the most important factors before and during the regeneration process is the type and concentration of cytokinin applied. Thidiazuron and benzyladenine are the most frequently used cytokinins in the regeneration systems, but their efficiency depends on genotype and other factors. Other cytokinins (e.g., zeatin and kinetin) have also been tested in several experiments and they were found in general to be less active. The organogenic ability of explants can also be increased by a properly selected cytokinin pre-treatment. Cytokinins applied in the pre-treatments can influence the leaf structure, which in turn can alter the regeneration capacity of the leaf explant. Interactions between factors of pre-treatments (hormones, light, and culture conditions) and factors of the regeneration phase should be considered. This review brings into focus the role of different cytokinins during in vitro shoot development, discussing their effects on the histology of leaves developed in vitro, and how this affects the subsequent regeneration process.

Main conclusion Studies on the tissue culture of apple have allowed for molecular, biotechnological and applied breeding research to advance. In the past 8 years, over 100 papers advancing basic biology, genetic transformation and cryobiology have emerged. Apple (Malus × domestica Borkh.; Rosaceae) is an important fruit crop grown mainly in temperate regions of the world. In vitro tissue culture is a biotechnological technique that has been used to genetically improve cultivars (scions) and rootstocks. This updated review presents a synthesis of findings related to the tissue culture of apple and other Malus spp. between 2010 and 2018. Increasingly complex molecular studies that are examining the apple genome, for example, in a bid to identify the cause of epigenetic mutations and the role of transposable elements in this process would benefit from genetically stable source material, which can be produced in vitro. Several notable or curious in vitro culture methods have been reported to improve shoot regeneration and induce the production of tetraploids in apple cultivars and rootstocks. Existing studies have revealed the molecular mechanism underlying the inhibition of adventitious roots by cytokinin. The use of the plant growth correction factor allows hypothetical shoot production from leaf-derived thin cell layers relative to conventional leaf explants to be determined. This updated review will allow novices and established researchers to advance apple and Malus biotechnology and breeding programs.