BACKGROUND:Optimization of biotechnological processes is traditionally limited by time-consuming trial-and-error approaches and the complexity of simultaneously optimizing multiple, often conflicting objectives. This applies particularly to plant tissue culture medium design, which therefore serves as the application case in this study. Recent advances in machine learning and evolutionary algorithms offer powerful alternatives, yet 80% of published studies rely on licensed software, and systematic data-driven optimization frameworks remain scarce. This creates significant barriers to adoption in both academic and commercial plant biotechnology. RESULTS:We introduce ADAM (Advanced Design and AI-Driven Modeling for Plant Tissue Culture Media Optimization), an open-access, web-based platform that transforms protocol development into a data-driven computational process. ADAM implements a complete ML-EA workflow through five integrated modules: 1. Design of Experiments (five different concepts) for systematic parameter exploration, 2. Data Preparation with automated quality control, and 3. Model Building using nine machine learning algorithms with automated selection. The platform enables Optimization (4.) through four advanced evolutionary algorithms (genetic algorithm, particle swarm optimization, NSGA-II, SMS-EMOA) for single- and multi-objective problems, with Evaluation (5.) tools to compare original versus optimized solutions. Validation across two plant tissue culture applications showed that ADAM's models matched or exceeded the predictive performance of manually optimized approaches in the original studies. The platform successfully identified multiple optimal culture conditions balancing conflicting objectives, providing experimentally testable predictions that reduce the trial-and-error cycle. CONCLUSIONS:Deployed as a browser-based application requiring neither specialized hardware nor software licenses, ADAM democratizes advanced AI optimization for plant biotechnology, eliminating traditional barriers to entry while maintaining the rigor and flexibility required for scientific research.

Apple replant disease (ARD) arises from repeated apple (Malus domestica) planting in the same area, disrupting physiological and morphological plant functions. Recent studies demonstrated that ARD occurs locally with low mobility in soil. The patchy distribution of ARD makes field identification of its severity difficult. Moreover, variability in soil properties can affect growth. Here, we aimed to identify drivers of small-scale growth variations with a pair-wise sampling approach at two ARD-affected orchards. We selected neighboring trees showing maximum differences in stem diameter growth. With this spatially explicit approach, large-scale heterogeneity in soil properties as a reason for differing growth was minimized. This design was applied to field plots differing in pre-cultures, i.e., comparing grass with Tagetes patula, the latter supposedly suppressing potential vectors of ARD. Various soil physical and chemical properties, the root phytoalexin content, and the bacterial and archaeal community composition were assessed. At one site, principal component analysis (PCA) separated neighbors with differing growth due to high particulate organic matter content, while no differences in soil physical properties, indicative of aeration differences or soil disturbances, could be detected. Elevated particulate organic matter content likely resulted from localized tree shredding. The worse-growing partners exhibited higher phytoalexin contents at the first site, which are general indicators of biotic stress and observed to increase in the presence of ARD. However, this was not associated with alterations of the rhizosphere bacterial and archaeal community composition as would be expected for ARD. At the second site, PCA showed no separation between tree groups, i.e., none of the measured variables could explain growth differences. Our work demonstrated that sampling neighboring trees with varying stem growth can identify co-occurring differences in related variables, some of which may reflect differences in ARD severity. Still, results were highly site-dependent and determined by the variables chosen for analysis.

This study presents the first reproducible vitrification-based cryopreservation protocol for shoot tips of sycamore maple (Acer pseudoplatanus L.), a tree of high ecological and economic significance in Central Europe. By safeguarding genetically verified material, including rare wood traits, this work supports the integration of the species into long-term ex situ conservation programmes. We investigated the influence of explant size, hardening duration, daytime temperature during hardening, and pre-culture across four genotypes. The protocol involved 4 wk of cold hardening under short-day conditions, a pre-culture at low temperature, vitrification with a plant vitrification solution, storage in liquid nitrogen, rapid rewarming, and gradual recovery under controlled light. Explant size proved decisive: shoot tips of 1–2 mm in length regrew at substantially higher rates and developed more vigorous shoots than larger explants. Four weeks of hardening with daytime temperatures between 3 and 7°C followed by pre-culture at 5°C produced the most reliable outcomes. The most responsive genotype reached regrowth in up to 90

Ri (root-inducing) technology, mediated by Rhizobium rhizogenes, presents a promising approach for modifying plant architecture. However, a comprehensive understanding of how complete, integrated wild-type T-DNA alters plant physiology is still lacking, as most related research has focused on partial gene sets and single species. To address this gap, our study undertakes the first systematic, comparative analysis of Ri genotypes across the phylogenetically distant species of rose (Rosa hybrida L.) and potato (Solanum tuberosum L.). We aimed to correlate organ-specific T-DNA gene expression with hormonal profiles and growth traits, thereby identifying both the general mechanisms of the Ri phenotype and species-specific differences. Morphological, molecular, and hormonal data were collected from three plant organs-leaves, stems, and roots-and analyzed across multiple Ri genotypes generated by R. rhizogenes strain ATCC 15834. All the Ri genotypes contained TL (left T-DNA) but differed in the presence of TR (right T-DNA) sequences. Compared with the respective wild-type plants, the Ri genotypes consistently presented shorter internodes (ratio of 0.5-0.89), in most cases smaller leaves (ratio of 0.52-1.03) and greater in vitro root formation (ratio of 0.71-2.34), as well as diverse expression levels of the rol and aux2 genes, a consistently higher concentration of cytokinins and altered levels of stress-related hormones in specific organs. Correlation and principal component analyses confirmed the relationships among rol gene expression, root number, and reduced shoot growth, whereas hormone responses were species-specific. Together, these findings complement the characteristics of typical Ri plants shown in previous studies and provide new insights into the underlying hormonal and genetic mechanisms, indicating altered stress signaling pathways in Ri genotypes. The first evaluations of root hair data in Ri plants contradict assumptions about a hairy root phenotype but also indicate an increased root tip diameter (ratio of 1.06-1.23) compared with that of the wild-type. Together, these findings underscore the importance of balancing architectural benefits with potential physiological trade-offs in the use of Ri technology for future breeding applications.

Apple replant disease (ARD) is a worldwide issue of complex biotic origin. The exact aetiology has yet to be fully elucidated and environmentally friendly and economically feasible countermeasures are sparse. The breeding of tolerant apple rootstock genotypes shows some promise as reduced sensitivity on selected ARD-affected soils has been shown for some rootstock genotypes. By adding insights into the root-associated microbiome, we aimed to complement previously published results, examining the response in growth, gene expression and production of phenolic compounds in roots of seven Malus genotypes in ARD soil. We selected the two least susceptible rootstock genotypes of the previous study, EMR.2 and G.935, in addition to the susceptible rootstock genotype M.26. The objective of this study was to gain insights into the bacterial community composition of the rhizosphere and root endosphere of less susceptible rootstock genotypes in comparison to the susceptible one. In an Illumina-based sequencing approach, the composition of rhizosphere as well as endophytic bacterial communities was investigated for apple plants in ARD soil and disinfected ARD soil. ARD impacted the rhizosphere stronger than the endosphere, where rootstock genotype influence was stronger. G.935 generally showed a more distinct and diverse microbiome than the other two rootstock genotypes. In bacterial communities, the relative abundance of Pseudomonadota decreased in ARD samples, while Actinomycetota increased. In the endosphere, this was mostly accounted for by Streptomyces. A qPCR approach confirmed the high abundance of Streptomyces. Interestingly, another qPCR approach did not indicate a high abundance of plant pathogenic Streptomyces in the same samples. ARD strongly influenced the bacterial microbiome in the rhizosphere and, to a lesser extent, the root endophytic microbiome. The distinct and more diverse bacterial microbiome of G.935 might help against biotic stress, such as ARD. However, EMR.2 was previously found to be less susceptible to ARD, too, suggesting additional factors must play a role and additional research is necessary. Pathogenicity of Streptomyces could not be proven but repeatedly high abundance of root endophytic Streptomyces under ARD conditions indicate an important role. The endophytic microbial communities of two East Malling rootstocks M.26 and EMR.2 are more similar to each other than to that of the Geneva rootstock G.935. Endophytic Streptomyces appear at high relative and absolute abundances in roots from ARD soil, regardless of rootstock genotype. qPCR results suggest that these endophytic Streptomyces are likely not plant pathogenic.

Apple replant disease (ARD) is a soil-borne disease that arises from replanting apple trees on land previously used for apple cultivation. There is interest in biomarkers that can reliably assess the severity of ARD by quantifying how strongly apple plants react to the disease in soils of different agro-environments. Thus far, transcriptomic studies of ARD-affected plants have examined only a few soils at a time, revealing that expression patterns vary among different soils. Here, we analyzed the expression of 90 candidate genes in the roots of apple plants (rootstock genotype 'M.26') grown in ARD-affected soils from 151 sites across Germany to test whether a consistent pattern of gene expression under ARD-conditions exists. Additionally, the expression of the candidate genes was analyzed in the leaves of apple plants grown in 18 different ARD-affected soils. Most of the genes (72) showed significantly upregulated expression in roots under ARD conditions, while only 11 showed significantly upregulated expression in leaves, suggesting that these genes play a significant role in the ARD reaction in roots but only a limited or no role in leaves. The candidate genes were evaluated for their potential as ARD biomarkers, defined by their consistently increased expression under ARD conditions across different soils and correlation with ARD severity. The accordingly selected ARD biomarker genes in roots include genes involved in phytoalexin biosynthesis, lignin metabolism, ethylene metabolism, cyanogenesis, detoxification, programmed-cell-death, and plant defense. These biomarkers have the potential to assess the severity of ARD and open up new possibilities for disease diagnosis.

Accurate identification of Salicornia species is a fundamental prerequisite for their potential usability and domestication. This study utilized a multifaceted methodological approach integrating morphological, cytogenetic, and molecular techniques to identify species from available European Salicornia sources. The following methods were compared: nuclear DNA content analysis; application of marker-based DNA barcoding via four common Salicornia markers; investigations of RNA topologies of these marker sequences by predicting theoretical secondary structures; utilization of diagnostic single-nucleotide polymorphism (SNP) positions within the external transcribed spacer (ETS) marker sequences for European Salicornia taxa; comparison of three promising microsatellite (SSR) markers regarding their ability to differentiate Salicornia subspecies; and evaluation of morphological data on habitus and flower characteristics utilizing a Salicornia identification key. The results demonstrate that ETS marker analysis offers reliable and cost-effective species determination, with SNP comparisons being more user friendly than phylogenetic trees are, and microsatellite markers can be differentiated down to the subspecies level via fragment length differences. However, microsatellite analysis alone is not suitable for primary species identification. DNA content can provide a rough estimation of potential species and is already more reliable than morphological methods. The differentiation among species is crucial for creating transparency for farmers and consumers and for initiating breeding processes, particularly within the context of frequent misidentification.

Microfluidic technologies offer powerful tools for miniaturized and highly controlled biological experiments, yet their application in plant research remains underexploited. In this study, we present a droplet-based microfluidic platform tailored for the encapsulation and cultivation of plant protoplasts, enabling long-term observation of cell development at nearly single-cell resolution. Protoplasts isolated from leaves of Nicotiana tabacum, Brassica juncea, and Kalanchoe daigremontiana were used to evaluate the platform’s suitability across diverse plant species. Our results demonstrate species-dependent responses to microfluidic cultivation, with tobacco protoplasts showing the highest viability. The system permits dynamic tracking of cell fate within individual droplets and supports the quantification of stochastic and concentration-dependent responses to chemical stimuli. Using tobacco protoplasts, we further investigated the effect of low concentrations of cytokinins (BAP) and auxins (NAA) for the early protoplast culture, up to the first division. Low concentrations (20–80 µg·L⁻¹) significantly enhanced cell survival and cell growth, while higher doses did not yield additional benefits. This work underscores the potential of droplet-based microfluidics as a high-resolution, low-volume platform for protoplast-based assays and dose-response screening, with applications across diverse plant biotechnology studies.

Sycamore maple (Acer pseudoplatanus L.) is an ecologically and economically important hardwood in Central Europe. For high-value genotypes, like wavy grain maple, in vitro propagation offers a promising tool for targeted multiplication and conservation. This study contributed to an optimised micropropagation protocol by testing disinfection protocols, silver nitrate supplementation, cytokinin type, and light conditions. Winter buds from 36 donor trees were disinfected with either sodium hypochlorite (NaOCl) or sodium dichloroisocyanurate (NaDCC). NaDCC markedly reduced contamination (5.2

The trace element selenium is essential for human nutrition but is distributed unevenly in soils worldwide with extensive selenium-deficient regions and selenium-enriched (seleniferous) areas. Neptunia amplexicaulis is one of the strongest selenium hyperaccumulator plants known and native to Australian seleniferous soils. Research in the genetic background of the selenium accumulation and tolerance mechanisms of this species lacks biotechnological and molecular tools for functional genetics. Therefore, this study aimed to develop a de novo shoot regeneration protocol for N. amplexicaulis and validate an selenium accumulation test system. Callus was induced on root and hypocotyl explants excised from 5-day old seedlings and cultured on an adjusted MS medium (SIM9) containing 4.5 µM Thidiazuron (TDZ) for two weeks in darkness. After this period, the TDZ concentration was reduced to 0.45 µM, and the explants were transferred to light conditions. In addition, seedlings of N. amplexicaulis, N. heliophila and Medicago truncatula were placed on vertical MS agar plates containing 1.5 mM (standard) or 0.1 mM (low) magnesium sulphate with 0, 30, 90 µM sodium selenate. Initial shoot differentiation was observed 6 weeks after culture initiation. This regeneration response was successfully repeated in a second experiment. The outgrow of the shoot buds into complete shoots was not yet achieved but requires additional media optimization. Additionally, spontaneous shoot regeneration from a root was observed, highlighting potential for further studies. In vitro grown seedlings demonstrated efficient, selective selenium uptake in N. amplexicaulis and identified M. truncatula as a secondary selenium accumulator with selenium concentrations of > 300 µg Se g−1 DM. This project presents the first protocol for inducing early stages of development of indirect shoot organogenesis in N. amplexicaulis from hypocotyl and root explants as prerequisite for genetic transformation, though completing the regeneration cycle remains challenging. Neptunia amplexicaulis hyperaccumulates selenium also under in vitro conditions.

Apple replant disease (ARD) is a problem in apple tree and fruit production worldwide and its etiology is not fully understood. This study aimed to evaluate the tolerance of six apple genotypes, including the susceptible rootstock 'M.26', three potentially tolerant rootstocks 'EMR.2', 'G.202' and 'G.935', and two genebank accessions Malus spectabilis MAL0130 and Malus sargentii MAL0739 in ARD soils from six sites. Using a greenhouse biotest, plants were grown for 56 days in ARD or the respective γ-irradiated soil. No genotype showed consistent lack of growth reduction in all ARD soils, indicating that no universal tolerance exists within the tested set of genotypes. Three genotypes ('M.26', MAL0130, MAL0739) were selected for analyses of ARD indicator gene expression and phytoalexin contents in roots as measures of ARD-response. Biphenyl synthase 3 expression and phytoalexin content strongly correlated with highest levels observed in roots of the susceptible rootstock 'M.26'. MAL0739 showed the lowest response, which was not consistently reflected in improved plant growth across all soils. Analysis of the bacterial and fungal communities in three root-affected ARD soils revealed soil- and genotype-specific differences. Bacterial alpha-diversity was higher in MAL0739 compared to 'M.26', while beta-diversity was mainly shaped by the soil. Significant enrichment of Nectria was observed for 'M.26' grown in one ARD soil. Phytoalexin contents and potentially plant beneficial microbial taxa correlated negatively. Our findings highlight that ARD tolerance in apple genotypes is linked to the soil microbiome and other soil properties, indicating the need for an integrated approach to manage ARD.

Plant growth-promoting bacteria (PGPB) can beneficially modulate rhizosphere microbial communities, potentially improving plant health and reducing disease incidence. Limited research exists on the influence of PGPB inoculation on the rhizosphere microbial communities of apple plants, particularly in soils affected by apple replant disease (ARD). Here, we evaluated the capacity of GFP-labelled Priestia megaterium B1 (designated as P. megaterium B1L5) to colonize the roots of apple plantlets grown in two soils: ARD-affected soil and ARD-unaffected grass soil. We investigated its influence on plant growth in ARD-affected soil and its potential to mitigate ARD-related symptoms. We also assessed how its inoculation modulates the rhizosphere microbial communities, with emphasis on changes that may support plant health, particularly in ARD-affected soils. P. megaterium B1L5 successfully colonized apple roots in both soils 6 days post-inoculation (dpi), but was not detectable at 33 dpi. In ARD-affected soil, plants inoculated with vegetative cells or spores displayed a lower proportion of blackened root tips compared to uninoculated controls. Beta diversity and PERMANOVA analyses demonstrated a significant influence of inoculation on the bacterial communities in both soils at 6 and 33 dpi (p = 0.001). Furthermore, inoculation enriched the rhizosphere of apple plantlets with potential plant-beneficial bacteria, such as Luteimonas, Lysobacter, Pseudomonas, Sphingomonas, Sphingobacterium, Rhodanobacter, Pedobacter and Flavobacterium. In contrast, fungal communities remained largely unaffected by inoculation. Most bacterial and fungal shifts observed in the rhizosphere of inoculated plantlets at 33 dpi did not exhibit similar patterns in uninoculated controls over time, indicating that these shifts were largely driven by the inoculum rather than by plant development or natural microbial succession. Our results highlight the capacity of P. megaterium B1L5’s to transiently colonize apple plant roots across different soil environments. The observed tendency toward reduced root tip blackening in inoculated plants grown in ARD-affected plants reflects its potential for alleviating stress associated with ARD. Additionally, inoculation with P. megaterium B1L5 promoted beneficial shifts in the rhizosphere microbiome by enriching bacterial taxa commonly linked to plant health. These findings indicate that P. megaterium B1L5 presents a candidate for ARD mitigation, however its long-term efficacy and practical application should be further evaluated.

Apple replant disease (ARD) represents a dysbiotic rhizosphere condition potentially driven by root exudates including phytoalexins at the root–soil interface. A promising mitigation strategy could be the application of bioinoculants that reduce these compounds and foster a diverse microbiome. This study investigated the effects of Rhodococcus pseudokoreensis R79T, a strain with benzoate-degrading capabilities and genetic potential to degrade biphenyls, on the rhizosphere microbiome of apple plantlets grown in ARD-affected soil in a greenhouse experiment. We applied R79T at 10⁶ to 10⁹ CFU/ml, assessing its impact on bacterial 16S rRNA diversity and abundance, as well as the abundance of biphenyl dioxygenase (bphd) genes. Eight weeks post-inoculation reads of strain R79T persisted in the rhizosphere, particularly at higher inoculation levels. Inoculation enhanced bacterial diversity and bphd gene abundance, with significant shifts in community composition. Key responders included members of Gaiellales, which increased, and Streptomyces, which decreased. Co-occurrence network analysis revealed that inoculation promoted positive interactions, more homogeneous connectivity, and a higher degree of connections. Effects on bacterial community structure varied significantly with inoculation concentration. The fact that R79T enhanced rhizosphere bacterial diversity and modulated community composition in ARD-affected soil highlights the potential of R79T to reshape microbial interactions. Further research is needed to elucidate the mechanisms underlying these effects, including studies on in situ degradation of phytoalexins and inoculation of R79T alongside bacteria for plant growth promotion (PGP) in synthetic communities for elevated efficiency against ARD.

Plant-soil feedback and soil microbial legacies play crucial roles in replanting success of apple. This study investigated how different soil amendment strategies influence these factors in replant disease-affected soil. Two approaches were tested: (i) the preculture and amendment of catch crops-either a single species, Tagetes patula, or a diverse catch crop mixture (CCM), and (ii) the inoculation of plant-beneficial microbes-bacteria, arbuscular mycorrhizal fungi, or their combination (SynC). Apple rootstock M.26 was grown for seven weeks in a greenhouse, and plant growth, soil nutrients, root phytoalexins, and microbial communities in rhizosphere and root-affected soil were analyzed. Catch crop amendments but not microbial inoculations, significantly altered soil nutrients. Root length increased significantly under CCM, and in tendency in Tagetes and SynC. Phytoalexin contents were lowest in Tagetes and highest in CCM, both differing from the control in specific compounds. Microbiome analysis revealed that catch crops strongly modulated fungal communities in rhizosphere and root-affected soil, favoring potentially beneficial Linnemannia and Mortierella, while microbial inoculations predominantly modulated bacterial/archaeal rhizosphere communities. Our results suggest that catch crops and microbial inoculants induced distinct shifts in soil-plant-microbe interactions under replanting conditions.

Apple replant disease (ARD) causes reduced growth and fruit yield and affects orchards and tree nurseries worldwide. A number of pathogens have been consistently identified as causal agents of ARD; however factors affecting disease-severity are not fully understood. We examined five soils from German tree nurseries and apple orchards featuring different soil characteristics and replant histories. We aimed to link the plant-soil interaction to replant disease severity. In a greenhouse experiment, young apple plants were grown for eight weeks on untreated and disinfected (control) soils. Growth parameters were recorded to evaluate the severity of ARD. The defence response of the plants was examined by expression analysis of ARD indicator genes (BIS3, B4H and ERF1B) and GC–MS-based detection of phytoalexins. The fungal and bacterial rhizosphere communities were investigated by ITS and 16S rRNA amplicon sequencing, respectively. After eight weeks, ARD symptoms were observed on all soils. Growth depression was highest on soils that had faced intensive apple cultivation and lowest on a soil with only one year of apple cultivation prior to the experiment. These results correlated with increases in the BIS3 expression level and the phytoalexin content in the roots. No bacteria and fungi commonly found in increased abundance in ARD soils were consistently detected in all soils. Replant history influenced disease severity more than soil characteristics. ARD symptoms correlated with BIS3 expression and phytoalexin (PA) formation. PA exudation increased the relative abundance of bacterial genera with the potential ability to degrade phenolic compounds.

Adventitious root (AR) formation is a genetically complex trait with high genotypic variability. Therefore, only a limited range of cultivars are currently propagated by cuttings in rose. In this study, we analysed the anatomy of in vitro shoots, the early formation of root primordia (RP) and the formation of ARs in a diverse set of 106 rose genotypes. Correlation analysis indicated that the growth in shoot diameter and the vasculature dimensions after 1 week of rooting contributed to successful AR formation. Using phenotypic data for genome-wide association studies (GWAS) analyses, nine significantly associated single nucleotide polymorphisms (SNPs) and genomic regions contributing to various RP and AR formation traits were identified. The contribution of genomic regions to trait variation was notably greater for traits associated with earlier processes than for traits associated with later developmental stages. The combination of RP and AR data allowed the detection of regions by GWAS that contain factors that potentially limit RP emergence. Homologues of 47 genes known to be involved in AR formation from the literature could be assigned to the identified peaks. Further studies are needed to investigate the suitability of SNPs exhibiting strong effects as allele-specific PCR markers for use in breeding.

IntroductionThe presence of wounds in addition to the excision-induced wounds after severance from the stock plants is known to positively influence adventitious root formation of woody plant cuttings. Previous morphological studies highlighted laser wounding as a technique allowing to precisely control the decisive ablation depth. However, the biochemical processes involved in the response of rooting to the additional wounding remained unexplored.MethodsThe present study analyzed changes in the plant hormone and carbohydrate profiles in response to laser treatments of rose leafy single-node stem cuttings (Rosa canina ‘Pfänder’). Concentrations of four groups of plant hormones and of carbohydrates were monitored in three different stem sections of the cutting base during the first eight days after excision of cuttings. In addition, histology was employed to investigate anatomical changes at the basal wound and the laser wounds at the start and the end of the experiment after 40 days.ResultsLaser ablation caused an increase of vascular tissue dimension directly in the laser wound, and increased the quantity and quality of rooting compared to control cuttings. A clear early local rise of jasmonic acid (JA) was detected directly in wounded areas after laser marking, as well as an increase in abscisic acid (ABA) that persisted for the subsequent days. Indole-3-acetic acid (IAA) levels were relatively high on day zero, but decreased thereafter. Interestingly, higher IAA levels were maintained in the stem section below the axillary bud compared with the opposite section. Laser-treated cuttings presented a clear increase in contents of IAA-amino acid conjugates (IAAGlu and IAAsp) and the oxidation product OxIAA. Differences in concentration of these IAA metabolites were related to the position of the laser wound relative to the axillary bud and leaf. Additionally, laser treatments caused gradually increased levels of the cytokinin N6-isopentenyladenine (iP) in laser-treated zones, and of zeatin riboside specifically when the laser wound was placed on the leaf-bud side. Additional laser wounding reduced starch and sucrose levels in all wounded sections at the end of the evaluation period, independently of the wounding location.DiscussionThe results of this study indicate that presence of additional injured tissue triggers a complex biochemical adjustment at the base of the cutting responsible of inducing vascular tissue growth and capable of generating a positive response to adventitious root formation.

This study explored the establishment and optimization of Ri (root inducing) technology for apple breeding, using the bacterium Rhizobium rhizogenes to obtain Ri lines with compact shoots and stronger root systems. The transformation and shoot regeneration for Malus domestica cultivars was studied in detail. Various R. rhizogenes strains, scion and rootstock genotypes, explant types, wounding methods and explant orientations were tested for hairy root induction. Most of the 16 tested strains, especially those with plasmid type III, induced hairy roots in the rootstock genotype ‘M26’. Although apple genotypes differed in response, in most of them roots were successfully induced using strains ATCC 15834, LMG 63 and LMG 150, with leaf blades outperforming petioles as explants. Wounding by scratching or sonication further improved transformation efficiency, as did placing leaf blades with their abaxial side upward on root induction medium. The majority (94%) of roots formed in one transformation experiment were tested PCR-positive for at least one T-DNA gene. Shoot regeneration experiments investigated salt concentrations, gelling agents, cytokinin types, concentrations, and a resting period on hormone-free medium. Shoot regeneration was highly genotype-dependent varying between 0 and 83%, whereas only minor, non-significant effects were observed for the treatments tested. Copy numbers of T-DNA genes were estimated using digital PCR for the first time in apple Ri lines. In the greenhouse, two Ri lines showed compact shoots and shorter leaves, but no enhanced root system. The improved protocol provides a valuable tool for breeders and scientists to obtain and further use Ri lines.

Multiple QTLs reveal the polygenic nature of R. rhizogenes-mediated transformation and hairy root formation in roses, with five key regions explaining 12.0–26.9

The production of apple fruits in orchards or plants in tree nurseries is negatively affected by apple replant disease (ARD), worldwide. Our objective was to develop a method to counteract ARD without applying chemical soil disinfection. We tested if an addition of clays with high release of plant available silicon reduces ARD symptoms and a biochemical effect of silicon on the plant defence reaction occurs. In a greenhouse experiment, apple rootstocks ‘M26’ were grown for 8 weeks in a sandy replant soil, a heat disinfected control and a grassland soil (Grass) with and without amendment by bentonite and the clay blend Florisol®TM Profi (6 and 18