Title Participants Abstract "Improved compact growth habit of Viola × wittrockiana through Rhizobium rhizogenes transformation" "Siel Desmet, Ellen De Keyser, Leen Leus, Johan Van Huylenbroeck, Danny Geelen, Emmy Dhooghe" "Compactness is an important breeding goal in the improvement of garden pansy varieties (Viola × wittrockiana Gams.). The desired compact stature is primarily obtained through the use of chemical growth retardants. Recently, an alternative strategy for creating compact ornamentals was proposed based on transformation by wild type rhizogenic agrobacteria and the regeneration of transformed hairy roots. Here, we report on the successful transformation and creation of compact garden pansy lines. The most efficient hairy root induction was obtained by Rhizobium rhizogenes strain ATCC15834. Shoot regeneration from hairy roots was obtained using 3 mg L−1 2iP and 0.3 mg L−1 2,4-D. The presence of R. rhizogenes T-DNA genes was confirmed in the regenerated lines. These lines displayed a varying degrees of dwarfed growth. Most notably the shoot length was reduced up to 49%. Other phenotypic traits that were induced include reduced peduncle length and smaller, wrinkled flowers. Several of these Ri lines with promising compactness have been selected for further breeding towards compact garden pansy varieties that are less dependent on chemical growth regulation." "DsRed reporter-gene based screening for evaluation of Rhizobium rhizogenes transformation efficiency" "Emmy Dhooghe, Siel Desmet, Johan Van Huylenbroeck, Ellen De Keyser" "An optimization of co-cultivation experiments with wild type Rhizobium rhizogenes strains consists of selecting the most optimal explant type, bacterial strain and plant genotype. Since Ri technology utilizes the native root inducing (Ri) plasmid of R. rhizogenes, the resulting hairy roots and hairy root regenerated plants do not contain any selectable marker nor reporter genes and are thus considered as non-GMO. However, phenotypic distinction between spontaneous root formation and true hairy roots is often unreliable, which encumbers the process of hairy root selection and subculture. This, combined with the fact that emergence of hairy roots can occur anywhere from 4 to 10 weeks post-inoculation, can make the development of an efficient hairy root induction protocol a long and arduous process. Here, we report on the opportunities and potential shortcomings of implementing a reporter-gene based strategy by DsRed for optimizing hairy root induction and selection in eight different herbaceous ornamental genera (encoded to keep this information confidential). Our results showed that, overall, the DsRed fluorescence gave an underestimation of the actual transformation efficiency, resulting in method accuracy of 66.7-100%. Except for Osteospermum fruticosum (3 cultivars) and genus E for which the method accuracy was only between 16.7 and 28.6 or 45.5%, respectively. The co-transformation efficiency was at minimum 66.8%. DsRed genes were only present when pRi genes were also monitored." "Hormone profiling of chrysanthemum hairy roots after co-cultivation with Rhizobium rhizogenes" "Ellen De Keyser, Jarek Tyburski, Natalia Mucha, Jacek Kesy, Tom Eeckhaut, Emmy Dhooghe" "We have used chrysanthemum as a model crop to determine auxin and cytokinin levels in hairy roots formed after coculture of leaf discs with Rhizobium rhizogenes. All 6 genotypes tested formed hairy roots, upon which the presence of T-DNA genes was tested with a qPCR assay, evaluating the presence of TL- as well as TR-genes. Transformation efficiencies varied between 14 and 100%, and for every cultivar, roots with either only TL-genes and roots with both TL- and TR-genes were found. 2-isopentenyladening (2iP) and indole-3-acetic acid (IAA) levels were compared for all 12 roots that yielded sufficient material for analysis. In two cultivars, multiple TL+TR positive independent root lines could be compared, showing a significant difference for as well auxin as cytokinin levels. However, both for 2iP and IAA, 10-fold differences were detected between roots induced on leaves of a single cultivar. This suggests that phytohormone levels, and probably subsequent hairy root regeneration, depend on individual pRi-gene integration for each separate hairy root." "Genetic and Morphological Variation of Belgian Cyperus esculentus L. Clonal Populations and Their Significance for Integrated Management" "No name available, Dirk Reheul, Jan De Riek, Ellen De Keyser, Benny De Cauwer" "Cyperus esculentus is an invasive troublesome neophyte in many arable crops across the globe. Analysis of the genetic and morphological profile of local C. esculentus clonal populations may be useful in explaining differential herbicide sensitivity found among distinct clonal populations and spatial distribution patterns. In this study, 35 Belgian C. esculentus clonal populations, evenly spread across the entire infestation area (30,689 km2) and covering a great diversity of farm and soil types, and hydrological and environmental conditions, were genetically characterized using amplified fragment length polymorphism (AFLP) profiling. These clonal populations were also grouped into different morphological clusters using data from shoot, tuber, and inflorescence number, fresh tuber biomass, individual fresh tuber weight, and number of germinable seeds collected in three consecutive years. Of the 271 AFLP markers, 207 were polymorphic. The gene diversity among clonal populations was 0.331 and three genetically distinct clusters were identified. Depending on observation year, clonal populations were grouped in four to five morphologically distinct clusters that closely aligned with the genetic clusters. The genetically distinct clusters differed in their geographical distribution pattern and range as well as in their morphological characteristics. Clonal populations belonging to clusters with broad distribution ranges produced numerous viable seeds. Clusters with clonal populations that produced large tubers were less widespread than the cluster with clonal populations producing many small tubers. The results suggest that tuber size, tuber number, and fecundity may all play an important role in the spread of C. esculentus. Morphotyping may be very useful in designing effective preventive and curative C. esculentus management strategies." "Antitranspirants putrescine and abscisic acid improve acclimatization of micropropagated spathiphyllum 'lima' regel" "Emmy Dhooghe, Peter Lootens, Christof Van Poucke, Ellen De Keyser, Johan Van Huylenbroeck" "A crucial phase for successful plant micropropagation is the transfer from lab to greenhouse. This acclimatization coincides often with plant losses. The possibility to improve acclimatization of Spathiphyllum plantlets were evaluated by the application of antitranspirants putrescine and abscisic acid (ABA) 3 days before transfer to ex vitro conditions. Different concentrations of putrescine (0, 0.882, 2.645, 4.408 and 8.815 g.l-1) and ABA (0, 0.5, 5 and 50 mg.l-1) were screened. For putrescine only 2.645 g.l-1 resulted in higher acclimatization rates. Overall ABA improved acclimatization significantly compared to the control treatment. No significant difference in the relative water content (RWC) was observed between control and the antitranspirants treatments. The impact at the physiological level of the best treatments (putrescine 2.645 g.l-1 and ABA 0.5 mg.l-1) were studied more into depth. Transpiration rate and stomatal conductance revealed that the most stressful days are the first days of the acclimatization phase. Antitranspirants had little significant impact on photosynthetic parameters, nevertheless ABA-treated plantlets showed less stress according to chlorophyll a fluorescence parameters. The endogenous ABA content increased during the first day of the acclimatization process. This research shows that antitranspirants can enhance acclimatization but the causality of their effectiveness is not unambiguous." "Compact shoot architecture of Osteospermum fruticosum transformed with Rhizobium rhizogenes" "Siel Desmet, Emmy Dhooghe, Ellen De Keyser, Johan Van Huylenbroeck, Danny Geelen" "Key message: Improved compact shoot architecture of Osteospermum fruticosum Ri lines obtained through Rhizobium rhizogenes transformation reduces the need for chemical growth retardants. Abstract: Compactness is for many ornamental crops an important commercial trait that is usually obtained through the application of growth retardants. Here, we have adopted a genetic strategy to introduce compactness in the perennial shrub Cape daisy (Osteospermum fruticosum Norl.). To this end, O. fruticosum was transformed using six different wild type Rhizobium rhizogenes strains. The most effective R. rhizogenes strains Arqua1 and ATCC15834 were used to create hairy root cultures from six Cape daisy genotypes. These root cultures were regenerated to produce transgenic Ri lines, which were analyzed for compactness. Ri lines displayed the characteristic Ri phenotype, i.e., reduced plant height, increased branching, shortened internodes, shortened peduncles, and smaller flowers. Evaluation of the Ri lines under commercial production conditions showed that similar compactness was obtained as the original Cape daisy genotypes treated with growth retardant. The results suggest that the use of chemical growth retardants may be omitted or reduced in commercial production systems of Cape daisy through implementation of Ri lines in future breeding programs." "Dehydrins and Soluble Sugars Involved in Cold Acclimationof Rosa wichurana and Rose Cultivar ‘Yesterday’" "Lin Ouyang, Leen Leus, Ellen De Keyser, Marie-Christine Van Labeke" "Rose is the most economically important ornamental plant. However, cold stress seriously affects the survival and regrowth of garden roses in northern regions. Cold acclimation was studied using two genotypes (Rosa wichurana and R. hybrida ‘Yesterday’) selected from a rose breeding program. During the winter season (November to April), the cold hardiness of stems, soluble sugar content, and expression of dehydrins and the related key genes in the soluble sugar metabolism were analyzed. ‘Yesterday’ is more cold-hardy and acclimated faster, reaching its maximum cold hardiness in December. R. wichurana is relatively less cold-hardy, only reaching its maximum cold hardiness in January after prolonged exposure to freezing temperatures. Dehydrin transcripts accumulated significantly during November–January in both genotypes. Soluble sugars are highly involved in cold acclimation, with sucrose and oligosaccharides significantly correlated with cold hardiness. Sucrose occupied the highest proportion of total soluble sugars in both genotypes. During November– January, downregulation of RhSUS was found in both genotypes, while upregulation of RhSPS was observed in ‘Yesterday’ and upregulation of RhINV2 was found in R. wichurana. Oligosaccharides accumulated from November to February and decreased to a significantly low level in April. RhRS6 had a significant upregulation in December in R. wichurana. This study provides insight into the cold acclimation mechanism of roses by combining transcription patterns with metabolite quantification." "The jasmonic acid pathway, rather than abscisic acid, may partly explain contrasting stomatal responses in two strawberry cultivars under osmotic stress" "Britt Merlaen, Ellen De Keyser, Marie-Christine Van Labeke" "Drought is a major threat in agriculture and horticulture, including commercial strawberry production. Here, we compare hormonal regulation of a first-line drought stress response, namely stomatal closure, in two Fragaria x ananassa cultivars, known to differ in their drought stress phenotype. We show that the observed difference in xylem abscisic acid accumulation cannot explain the different stomatal responses under osmotic stress. Foliar abscisic acid accumulation cannot fully account for the stomatal behavior in one of both cultivars either. An indirect effect of abscisic acid on stomatal conductance via an impact on leaf hydraulic conductance, possibly mediated via aquaporins, as is recently proposed in literature, was not observed here. Next, we show that these two cultivars respond differently to jasmonic acid and one of its precursors. This difference in sensitivity of the jasmonates pathway between both cultivars may partly explain the different stomatal response. This study contributes to the understanding of the regulation of an important drought stress response in an economically important crop prone to water deficit stress." "Cold Acclimation and Deacclimation of Two Garden Rose Cultivars Under Controlled Daylength and Temperature" "Lin Ouyang, Leen Leus, Ellen De Keyser, Marie-Christine Van Labeke" "Low temperature stress is an important abiotic stress for garden roses in northern regions. Two garden rose cultivars (‘Dagmar Hastrup’ and ‘Chandos Beauty’) were selected to study the role of dehydrin and of carbohydrate metabolism during cold acclimation and deacclimation under the controlled daylength and temperature. The presence of bud dormancy was also observed as this could prevent budburst during warm spells. Both cultivars showed a similar changing pattern of cold acclimation and deacclimation and did not differ in their lowest LT50 values. Dehydrin (RhDHN5) was up-regulated by low temperatures and not by dehydration stress as the stem water content remained stable during the treatments. Total soluble sugars accumulated with a transient up-regulation of RhBAM3 (a key gene in starch hydrolysis) for ‘Dagmar Hastrup’ at 2°C and a strong expression under both 2 and −3°C for ‘Chandos Beauty’. At 2 and −3°C, raffinose and stachyose strongly accumulated though the up-regulation of RhRS6 and RhGK differed in the cultivars. Although similar cold hardiness levels were reached, carbohydrate metabolism in response to cold stress is different in the two cultivars. Increasing the temperature after a cold period resulted in fast deacclimation as found by the downregulation of RhDHN5 and RhBAM3, the decrease of raffinose and stachyose. Bud endodormancy was hardly present in both cultivars." "Rhizogenic agrobacteria as an innovative tool for plant breeding: current achievements and limitations" "Siel Desmet, Emmy Dhooghe, Ellen De Keyser, Johan Van Huylenbroeck, Renate Mueller, Danny Geelen, Henrik Lutken" "Compact plant growth is an economically important trait for many crops. In practice, compactness is frequently obtained byapplying chemical plant growth regulators. In view of sustainable and environmental-friendly plant production, the search forviable alternatives is a priority for breeders. Co-cultivation and natural transformation using rhizogenic agrobacteria result inmorphological alterations which together compose the Ri phenotype. This phenotype is known to exhibit a more compact planthabit, besides other features. In this review, we highlight the use of rhizogenic agrobacteria and the Ri phenotype with regard tosustainable plant production and plant breeding. An overviewof described Ri lines and current breeding applications is presented.The potential of Ri lines as pre-breeding material is discussed from both a practical and legal point of view."