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.

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.

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.

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.

No abstract

No abstract

Endogenous carbohydrate (fructose, glucose and sucrose) fractions were measured in calli of potato genotypes with different field tolerance to drought. Under in vitro stress conditions induced by 0.8 M mannitol, sucrose level of calli increased extremely in medium-tolerant (by 424.5%) and sensitive (by 302.7%) genotypes whilst the rate of increase was 12-18-times lower in the drought tolerant variety. Results suggest the applicability of sucrose as biochemical marker for distinguish drought tolerant genotypes from great population in callus culture.

In vitro shoot multiplication responses of Amelanchier canadensis ‘Rainbow Pillar’ were studied on media solidifi ed with different gelling agents. The media were gelled either with 6.8 g l-1 fi brous agar-agar, or 50.0 g l-1 wheat starch, or 20.0 g l-1 Guar gum, or 15 g l-1 Isubgol or 50.0 g l-1 wheat starch mixed with 0.5 g l-1 Phytagel. Shoot cultures were grown for two months, thereafter the multiplication rates (number of newly developed shoots per explant) were counted and the length of shoots were measured. We found that the highest shoot multiplication of Amelanchier canadensis ‘Rainbow Pillar’ occurred on media gelled with Guar gum, while the longest shoots developed on media with Starch. About four-fold shoot number were obtained on media with Guar gum compared to the weakest results found on media gelled with Isubgol. Finally, considering all factors (shoot growth parameters, costs) the most economical gelling agent for Amelanchier canadensis ‘Rainbow Pillar’ was proved to be wheat starch among the tested alternatives which allows a 75.6% cost reduction.