Up until today, apple sport mutants proved to be indistinguishable from each other and their progenitors at the molecular level using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) marker techniques. This is not surprising, since the genomes of these somatic mutants differ... only in one or a few small regions that affect economically important characteristics, such as improved fruit colour, size, or flavour. In most cases, these genome differences are probably caused by retrotransposons which are able to convert their RNA transcripts to DNA with reverse transcriptase enzyme prior to reinsertion, but unable to leave the genome and infect other cells. Retrotransposon insertions can alter the expression of other genes and/or the structure of encoded proteins. The sequence-specific amplified polymorphism (S-SAP) technique is capable of revealing the genetic distribution of retrotransposable elements over the whole genome. The present study used this approach to try to characterize and distinguish 'Jonathan' somatic mutants via fingerprinting, which is an unsolved problem.
Altogether 40, mainly old Hungarian apple varieties were screened with six previously described microsatellite markers. A total of 71 polymorphic alleles were detected (average 11.8 alleles/locus) and the heterozygosity of markers averaged very high (0.8). The genetic variability among the genotypes proved to be so remarkable that as few as thr...ee markers from the applied six were enough to distinguish between the 40 varieties. This was also confirmed by the cumulative probability of obtaining identical allele patterns for two randomly chosen apple genotypes for all loci, which value was quite low: 2.53 x 10-5. The molecular identification of these genetically very different old apple genotypes could be very useful in future breeding programs.
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 ran...ge 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.
A detailed examination of the production of ethylene and other ripening parameters during storage period has been undertaken in transgenic apple fruits, where the ethylene biosynthesis was inhibited by antisense ACS (l-aminocyclopropane-l-carboxylate synthase) gene. Data indicate down regulation of ethylene production, softening and spoilage in... some transgenic lines. In some cases ethylene production was inhibited for over 90 percent, considerable reduction of softening and spoilage was observed probably due to the reduced activity of cell wall degradable enzymes. ACS activity was also monitored during ripening. The fruits of the best transgenic lines could be stored for minimum 4-5 months longer under 5 °C cold room storage conditions and one month longer at normal room temperature. This molecular approach can provide an alternative way to replace the commonly used and costly atmospheric storage of fruits.
Dianthus chinensis and Dianthus caryophyllus varieties were tested for shoot regeneration from leaf and petal explants and transformed with Agrobacterium tuniefaciens strains (EHA 105 and LBA 4404) harbouring an apple derived ACS cDNA in antisense orientation in order to reduce ethylene production and influence the et...hylene dependant traits in carnation. After transformation regenerating shoots were selected on MS medium containing 50-75-100-125-150 mg/1 kanamycin and supplemented with 1 mg/1 BA, 0.2 mg/1 NAA. Transgene integration was proved by PCR analysis with npt II spcific primers followed by Southern hybridisation of DNA isolated from green shoots on medium containing 150 mg/1 kanamycin. Several putative transformants were subjected to RT-PCR in order to examine the npt 11 expression at mRNA level. Both the transformant and the non-transformant plants were potted into glasshouse to observe the effect of changed ethylene production on flowering time, petal senescence and vase life.