The effects of a new type of aromatic cytokinin, meta-topolin, on the morphology and histology of apple leaves and its post-effects on the subsequent shoot regeneration from in vitro leaves were studied in cv. Royal Gala. The media applied for pre-treatment differed from each other in their cytokinin composition: medium No. 1 contained no cytokinin, No. 2 was supplemented with 0.5 mg l-1 benzyladenine, while Nos. 3-6 contained meta-topolin, the new type of cytokinin, in four concentrations (0.5-1.0-1.5-2.0 mg l-1). After a 3-week pre-treatment on these media shoot regeneration was induced on two test regeneration media containing thidiazuron (0.2 mg l-1) or benzyladenine (5.0 mg l-1). Irrespective of the pre-treatments, high regeneration (97-100%) was observed on all the regeneration media. however, the conditioning of apple shoots for three weeks on medium supplemented with meta-topolin in a concentration range between 0.5 and 1.5 mg l-1 caused a significant decrease in the rate of vitrified shoots (down to 13.4%) and increased the number of regenerated shoots per leaf segment significantly (up to 15.1). There was a positive correlation between the histological status and regeneration capacity of in vitro leaves. According to these results, meta-topolin, as a new source of cytokinin, could increase the morphogenic potential of apple leaves.

The requirements for in vitro micrografting in apple are described. In vitro multiplicated shoots of cv. Royal Gala were the sources of rootstocks and scions after different pre-treatment, respectively. Oxidative browning of cut surfaces could be inhibited by the use of antioxidant mixture during grafting process. Scion base cut in v-shape was stuck by 1% agar-agar solution into the vertical slit of rootstock. There was no any displacement and the rate of fused and further developed grafts was 95 percent. Agar-agar between the rootstock and scion made the transport of different materials possible and hold the graft units together until the fusion took place. Fusion was proved also by histological studies. Some of in vitro micrografts were planted and acclimatisated and the survival was 100 percent.

The effects of different types of cytokinins on the shoot regeneration from leaf explants of apple scion 'Royal Gala' and apple rootstock 'M.26' were evaluated. Regeneration media contained either thidiazuron, or 6-benzylaminopurine, or meta-topolin, or zeatin, or kinetin, or their N9-ribosides, respectively, in the concentration range 0.5 to 8.0 mg 1-1. Effects of 'these cytokinins were evaluated on the percentage of regeneration (R%) and that of vitrification (V%) and on the number of regenerated shoots per explant (SN). Organogenetic index (0I) calculated from these data was used for the evaluation of efficacy of cytokinins. The course of shoot organogenesis also was followed using stereomicroscope. Types and concentrations of cytokinins applied in the regeneration media influenced each parameter significantly and the regeneration answer was strongly genotype-dependent. The best regeneration (SN: 11.08, 01: 7.5) was achieved in `Royal Gala' by using TDZ in concentration of 0.5 mg 1-1 (2.271,1M). There was a clear relationship between the effect on the regeneration efficacy and the chemical structure of cytokinins considering classical cytokinins, namely N9-ribosides applied in less concentration than non­ribosides have the same or best regeneration effects except for 6-benzylaminopurine riboside. However, similar relationship could not be detected in the case of 'M.26'. SN was the highest (3.22) using 6.5 mg 1-1 (18.2011M) 6-benzylaminopurine riboside or 8.0 mg 1-1 (21.44 µM) meta-topolin riboside (3.18). SN was not significantly lower (3.12) by using 2.0 mg 1-1 (9.08 1M) TDZ, however, OI was about half as big (0.63 compared to 1.29 or 1.74 with 6-benzylaminopurine riboside or meta-topolin riboside, respectively). 'Royal Gala' had higher organogenetic ability, than `M.26': 3.5-fold higher shoot number per explant and more than 4-fold higher organogenetic index was reached with this cultivar than with 'M.26'. Moreover, the similar developmental stage of shoots could be observed 3-5 days earlier than in 'M.26' and if explants of 'Royal Gala' were further cultured with 3 weeks, SN increased from 11.08 to 24.42 on TDZ-containing regeneration medium, which might suggest higher organogenetic ability, too.

The effects of different aromatic cytokinins applied in different concentrations and combinations were investigated on the histology of in vitro apple leaves and their post-effects on subsequent shoot regeneration from these leaves were studied. Great differences in the anatomical structure of leaves could be detected originating from media containing different types and concentrations of aromatic cytokinins. The number of regenerated shoots per explant and the organogenetic index were used for the evaluation of the post-effect of aromatic cytokinins on shoot regeneration. The histological structure of leaves used for regeneration and their regeneration response showed a good correlation. When the pre-treatment caused a juvenile-like or less-differentiated structure, the number of regenerated shoots per explant increased and often vitrification also decreased and consequently the organogenetic index also increased. A strong interaction between cytokinin-content (type and concentration) of the pre-treatment medium and that of the regeneration medium could also be detected.

Micrografting was used in our experiments for establishment of in vitro culture from one rootstock (`JTE-F') and three scion cultivars (`Remo', 'Rewena' and `Reanda') of apple. Shoot tips of these cultivars were harvested from field and grafted onto in vitro rootstock cultivars. Their survival and development were studied. 42-93% of shoot tips survived and developed further depending on cultivar. Impermanent browning of sticking agar-agar could be observed in 21-25% of the micrografts depending on cultivars but discolouration of agar-agar ceased within one week and did not cause any death of shoot tips. We used micrografting successfully for establishment of in vitro culture from cultivars, from which earlier with conventional methods the culture establishment was not possible because of hard tissue browning. However, further studies are necessary to ensure the survival and development of shoots after removing them from micrografts.