Plant memory is an adaptive mechanism that plants can use to increase their fitness and cope with adverse environmental stresses. In this study, mRNA-sequencing (mRNA-seq), whole-genome bisulfite sequencing (WGBS) and real-time quantitative PCR (RT-qPCR) methods were applied for evaluating formation and maintenance of somatic transcriptional memory after treatment with ultrasound and drought stimuli in tomatoes. In addition, the effects of repeated stimuli, as well as the association-forming ability of plants were studied when they were trained previously with combined stimuli. Two days after exposure to the two stimuli applied alone or in combination, significantly altered gene transcription and DNA methylation were revealed. Using four selected target genes, we demonstrated that plants memorized stimuli for 5-10 d, in a gene- and stimulus-dependent way. The repeated application of the stimuli caused various alterations in gene transcription behavior, such as habituation, sustained induction or modified reinduction. Plants were able to use one conditioned stimulus as a predictor of the other, unconditioned one, after conditioning in the case of 3 out of 4 target genes, and used their transcriptional memory associatively. The exploitation of plant memory and associative learning may contribute to the development of new strategies to increase plant stress resilience.

Exogenous cytokinin supply is a crucial factor during the in vitro shoot multiplication of apples. Meta-topolin has been shown to cause improved multiplication rate, higher quality in vitro shoots with better rooting, and acclimatization ability than the widely used benzyl adenine. The effects of benzyl adenine and meta-topolin on mRNA transcription in in vitro shoots were analyzed by using mRNA-seq, bioinformatics analysis, GO annotation, and KEGG mapping. The present investigations revealed that there were about 6-fold more significantly up-, or down-regulated genes (DEGs) in shoots grown on the benzyl adenine-containing medium than in those grown on the meta-topolin-containing medium. DEG analyses showed that WRKYs, bHLH, and MYB were the most affected transcription factors after both cytokinin treatments, while the expression of MIKC-type MADS-box, ERF, and AP2 transcription factors changed only after benzyl adenine treatment. DEGs related to auxin transport and signaling, as well as auxin synthesis, were differently affected by the two cytokinins. The DEG encoding cytokinin hydroxylase-like protein and related to trans-zeatin biosynthesis was up-regulated only after benzyl adenine treatment. The DEG encoding gibberellin 20 oxidase 2-like was down-regulated after a benzyl adenine supply while it was up-regulated after a meta-topolin supply. Changes in the cytokinin–auxin balance and gibberellin biosynthesis in in vitro shoots may contribute to the morphological differences previously observed for the two cytokinins.

One of the most critical factors in maintaining healthy in vitro axillary apple shoot cultures is the cytokinin content of the medium. The effects of two cytokinins applied in the medium, thidiazuron and 6-benzylaminopurine riboside, each applied in a quantity of 4.5 µM, were studied on mRNA transcription of in vitro apple shoots. Transcriptomic response of in vitro apple shoots was investigated at two time points; at 48 h and 4 weeks (at the end of the subculture) after the cytokinin application by mRNA-seq, bioinformatics analysis, GO annotation and KEGG mapping. This study revealed that different expression intensities of DEGs were related to TF families (mainly WRKY, MYB, AP2/ERF, bHLH) associated with plant growth and development, in addition to enzymes involved in genetic and environmental information processing, cell motility, the zeatin biosynthesis, the tryptophan metabolism, the phenylpropanoid biosynthesis, and cutin, suberin, and wax biosynthesis. A putative roadmap based on changes in gene expression intensity that may be in association with various in vitro apple shoot development and growth disorders caused by different cytokinin supplies was conceived.

The application of dual cytokinins can significantly enhance shoot multiplication rates in specific apple cultivars compared to standard protocols using a single cytokinin. This study presents a comprehensive analysis of shoot multiplication parameters and the underlying transcriptomic response of two distinct apple scion cultivars, cvs. Húsvéti rozmaring and McIntosh, to the simultaneous application of two cytokinins (BA and KIN). Morphological parameters were recorded, followed by comparative RNA-seq analysis and RT-qPCR validation. Our results demonstrate that the BA+KIN treatment induces a unique transcriptomic signature in both cultivars, which cannot be explained by a simple dose–response effect. In cv. McIntosh, 76% of the DEGs were uniquely regulated by the combination, while in cv. Húsvéti rozmaring, although the overlap with single treatments was higher, 17% of the DEGs (representing 1218 genes) were still exclusively activated by the BA+KIN treatment. The fact that the combined treatment recruits specific gene sets and metabolic pathways that remain silent under single BA or KIN applications—regardless of the cultivar—strongly supports a synergistic or non-additive hormonal interaction rather than a response to increased total cytokinin concentration. The dual treatment revealed 3209 DEGs in the inter-cultivar comparison, reflecting distinct strategies: cv. Húsvéti rozmaring achieved high efficiency growth by down-regulating internal hormones, whereas cv. McIntosh exploited intense auxin signaling and hormonal plasticity to maximize bud release. These results prove that distinct molecular pathways can lead to peak performance depending on the apple cultivar.