Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus.

Industrial chicory (Cichorium intybus var. sativum) and witloof (C. intybus var. foliosum) are crops with an important economic value, mainly cultivated for inulin production and as a leafy vegetable, respectively. Both crops are rich in nutritionally relevant specialized metabolites with beneficial effects for human health. However, their bitter taste, caused by the sesquiterpene lactones (SLs) produced in leaves and taproot, limits wider applications in the food industry. Changing the bitterness would thus create new opportunities with a great economic impact. Known genes encoding enzymes involved in the SL biosynthetic pathway are GERMACRENE A SYNTHASE (GAS), GERMACRENE A OXIDASE (GAO), COSTUNOLIDE SYNTHASE (COS) and KAUNIOLIDE SYNTHASE (KLS). In this study, we integrated genome and transcriptome mining to further unravel SL biosynthesis. We found that C. intybus SL biosynthesis is controlled by the phytohormone methyl jasmonate (MeJA). Gene family annotation and MeJA inducibility enabled the pinpointing of candidate genes related with the SL biosynthetic pathway. We specifically focused on members of subclade CYP71 of the cytochrome P450 family. We verified the biochemical activity of 14 C. intybus CYP71 enzymes transiently produced in Nicotiana benthamiana and identified several functional paralogs for each of the GAO, COS and KLS genes, pointing to redundancy in and robustness of the SL biosynthetic pathway. Gene functionality was further analyzed using CRISPR/Cas9 genome editing in C. intybus. Metabolite profiling of mutant C. intybus lines demonstrated a successful reduction in SL metabolite production. Together, this study increases our insights into the C. intybus SL biosynthetic pathway and paves the way for the engineering of C. intybus bitterness.

Assessment of transcriptional reprogramming of lettuce roots in response to chitin soil amendment.

Chitin soil amendment is known to improve soil quality, plant growth and stress resilience, but the underlying mechanisms are not well understood. In this study, we monitored chitin's effect on lettuce physiology every two weeks through an eight-week growth period, analyzed the early transcriptional reprogramming and related metabolomic changes of lettuce, in response to crab chitin treatment in peat-based potting soil. In commercial growth conditions, chitin amendment still promoted lettuce growth, increased chlorophyll content, the number of leaves and crop head weight from week six. The flavonoid content in lettuce leaves was altered as well, showing an increase at week two but a decrease from week six. Transcriptomic analysis showed that over 300 genes in lettuce root were significantly differentially expressed after chitin soil treatment. Gene Ontology-term (GO) enrichment analysis revealed statistical overrepresentation of GO terms linked to photosynthesis, pigment metabolic process and phenylpropanoid metabolic process. Further analysis of the differentially expressed genes (DEGs) showed that the flavonoid pathway was mostly upregulated whereas the bifurcation of upstream phenylpropanoid pathway towards lignin biosynthesis was mostly downregulated. Metabolomic analysis revealed the upregulation of salicylic acid, chlorogenic acid, ferulic acid, and p-coumaric acid in chitin-treated lettuce seedlings. These phenolic compounds (PCs) mainly influence the phenylpropanoid biosynthesis pathway and may play important roles in plant defense reactions. Our results suggest that chitin soil amendments might activate induced resistance by priming lettuce plants and promote lettuce growth via transcriptional changes.

Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators.

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

Compact shoot architecture of Osteospermum fruticosum transformed with Rhizobium rhizogenes.

KEY MESSAGE: Improved compact shoot architecture of Osteospermum fruticosum Ri lines obtained through Rhizobium rhizogenes transformation reduces the need for chemical growth retardants. 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.

An asymmetric protoplast fusion and screening method for generating celeriac cybrids.

Celeriac F1 hybrid seed production is currently complicated due to the instability of cytoplasmic male sterile lines. To develop alternative alloplasmic CMS lines, an asymmetric protoplast fusion and hybrid screening methodology was established. Celeriac suspension cells protoplasts were used as the acceptor and carrot, coriander and white celery mesophyll protoplasts as the donor for protoplast fusion experiments. Acceptor cytoplasmic inheritance was inhibited by iodoacetamide treatment and donor nuclear genome inheritance was prevented by UV exposure. Protoplasts were selectively stained and fused using electroporation and polyethylene glycol, and candidate hybrid shoots were obtained. One chloroplast and three mitochondrial markers that could distinguish acceptor and donors organelles were used to characterize over 600 plants obtained after fusion events, without identifying any cybrid. In order to increase the testing efficiency a high number of micro plantlets were pooled and hence the presence of the carrot specific Atp1 marker in one of the pooled samples was detected. We demonstrated that fusion took place between celeriac and a carrot indicating that the creation of viable hybrids is possible although at a very low frequency. These findings open the path for new cytoplasmic hybridization and the isolation of novel CMS lines of celeriac.

Segregation of rol Genes in Two Generations of Sinningia speciosa Engineered Through Wild Type Rhizobium rhizogenes.

Rhizobium rhizogenes infects and transforms a wide range of plant species. It thereby introduces new genes located on transfer-DNA of the root inducing plasmid (pRi) into the plant genome and one of its abilities is to alter the host root system. Explants from pRi transformed roots from Sinningia speciosa were regenerated to create naturally transgenic Ri lines. The presence of rol and aux genes in the Ri lines was linked with altered growth characteristics: shorter peduncles, wrinkled leaves, delayed flowering and enhanced root growth. The potential of Ri lines for breeding was evaluated through consecutive backcrossing with the original host genotype. The progeny of reciprocal crosses showed non-Mendelian inheritance suggesting partial transmission of the of the aux and rol genes. The typical Ri phenotype observed in the primary Ri line was partially inherited. These results revealed that the Ri phenotype is a complex trait influenced by the genetic background of the Ri line.

Cold Acclimation and Deacclimation of Two Garden Rose Cultivars Under Controlled Daylength and Temperature.

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.

The jasmonic acid pathway, rather than abscisic acid, may partly explain contrasting stomatal responses in two strawberry cultivars under osmotic stress.

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.

Rhizogenic agrobacteria as an innovative tool for plant breeding: current achievements and limitations.

Compact plant growth is an economically important trait for many crops. In practice, compactness is frequently obtained by applying chemical plant growth regulators. In view of sustainable and environmental-friendly plant production, the search for viable alternatives is a priority for breeders. Co-cultivation and natural transformation using rhizogenic agrobacteria result in morphological alterations which together compose the Ri phenotype. This phenotype is known to exhibit a more compact plant habit, besides other features. In this review, we highlight the use of rhizogenic agrobacteria and the Ri phenotype with regard to sustainable plant production and plant breeding. An overview of 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.

Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum).

Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof.

A General Protocol for Accurate Gene Expression Analysis in Plants.

Gene expression analysis by means of RT-qPCR is a highly sensitive technique. However, this requires an accurate protocol for the whole procedure from sampling to data analysis. We have optimized this protocol specifically for the analysis of plant tissues. Special attention is paid to RNA quality and integrity and to the appropriate setup of the assays in order to be compliant with the MIQE guidelines. This protocol was already successfully applied in ten different plant species.

Physiological responses and aquaporin expression upon drought and osmotic stress in a conservative vs prodigal Fragaria x ananassa cultivar.

In order to improve the understanding of plant water relations under drought stress, the water use behavior of two Fragaria x ananassa Duch. cultivars, contrasting in their drought stress phenotype, is identified. Under drought, stomatal closure is gradual in Figaro. Based on this, we associate Figaro with conservative water use behavior. Contrarily, drought stress causes a sudden and steep decrease in stomatal conductance in Flair, leading to the identification of Flair as a prodigal water use behavior cultivar. Responses to progressive drought on the one hand and an osmotic shock on the other hand are compared between these two cultivars. Tonoplast intrinsic protein mRNA levels are shown to be upregulated under progressive drought in the roots of Figaro only. Otherwise, aquaporin expression upon drought or osmotic stress is similar between both cultivars, i.e. plasma membrane intrinsic proteins are downregulated under progressive drought in leaves and under short term osmotic shock in roots. In response to osmotic shock, root hydraulic conductivity did not change significantly and stomatal closure is equal in both cultivars. De novo abscisic acid biosynthesis is upregulated in the roots of both cultivars under progressive drought.

Differential efficiency of wild type rhizogenic strains for rol gene transformation of plants.

Rhizogenic agrobacteria induce extensive root proliferation, in several economically valuable, dicotyledonous plant species, a phenomenon referred to as "hairy roots." Besides their pathogenic nature, agrobacteria have proven to be a valuable asset in biotechnology and molecular plant breeding. To assess the potential of frequently used rhizogenic strains, growth in yeast extract glucose broth and antibiotic resistance was analyzed. Growth curves were established for Arqua1, NCPPB2659, LMG150, LMG152, and ATCC15834; and regression analysis of the exponential growth phase resulted in a reliable and standardized method for preparation of a bacterial suspension for inoculation. Cell density did not correlate with the timing of hairy root emergence. The highest number of hairy roots was obtained with an inoculum of 1 × 108 CFU ml-1 for Arqua1, NCPPB2659, and LMG152. Cell density of ATCC15834 did not affect the number of hairy roots formed. The identity of the rhizogenic strains for plant transformation was verified in phylogenetic analysis using average nucleotide identity (ANI), which also provided insight in their genetic diversity within the Rhizobium taxon.

Do New Breeding Techniques in Ornamentals and Fruits Lead to Essentially Derived Varieties?

Do new breeding techniques (NBT) lead to essentially derived varieties (EDV)? It depends! It depends on the definition of EDV in the plant variety right (PVR) laws and their interpretation by the courts. This paper aims at providing an overview of the EDV concept and an analysis of the question whether NBT lead to EDV on the basis of the UPOV 1991 Act, the most recent UPOV Explanatory Notes on EDV of 2017 as well as some selected PVR laws. Almost 30 years ago, the concept of EDV has been incorporated into the UPOV 1991 Act. In order to strengthen the rights of breeders, in particular to provide breeders of original genotypes an additional source of remuneration, a system of "Plant Variety Right specific dependency," based on "essential derivation," was developed. Only a very limited number of court cases have been concerned with EDV. However, an escalation in EDV-related conflicts can be expected in the future due to increased competition in the ornamental and fruit breeding business as well as to the application of more sophisticated NBT.

Seasonal changes in cold hardiness and carbohydrate metabolism in four garden rose cultivars.

We studied metabolic adaptations to cold stress in roses and identified genes in the carbohydrate pathway during acclimation and deacclimation. A field experiment with four rose cultivars belonging to different USDA plant hardiness zones was set up in Melle, Belgium (51° 0' N, 3° 48' E). The more cold-hardy cultivars ('Dagmar Hastrup' and 'John Cabot') reached their lowest LT50 value in December, indicating a rapid acclimation after the first occurrence of frost. Less cold-hardy cultivars ('Abraham Darby' and 'Chandos Beauty') reached their lowest LT50 in January/February when exposed to prolonged freezing temperatures. A cell dehydration pattern was found in the less cold-hardy cultivars 'Abraham Darby' and 'Chandos Beauty'. The expression of dehydrins (RhDHN5 and RhDNH6) was up-regulated during November-January. Carbohydrate metabolism is highly involved in cold acclimation in roses. Starch decreased from November towards January in all four cultivars and the hydrolysis of starch by the ß-amylolytic pathway (BAM, DPE2) was identified in 'Dagmar Hastrup' from November to January. Oligosaccharides correlated with cold hardiness in three cultivars although no significant upregulation in RhMIPS and RhRS6, key genes in their biosynthesis, was found. Higher sucrose levels were found during acclimation in hardy cultivars, although transcript levels of RhINV2 was more prominent in 'Chandos Beauty'.

Identification and substrate prediction of new Fragaria x ananassa aquaporins and expression in different tissues and during strawberry fruit development.

The newly identified aquaporin coding sequences presented here pave the way for further insights into the plant-water relations in the commercial strawberry (Fragaria x ananassa). Aquaporins are water channel proteins that allow water to cross (intra)cellular membranes. In Fragaria x ananassa, few of them have been identified hitherto, hampering the exploration of the water transport regulation at cellular level. Here, we present new aquaporin coding sequences belonging to different subclasses: plasma membrane intrinsic proteins subtype 1 and subtype 2 (PIP1 and PIP2) and tonoplast intrinsic proteins (TIP). The classification is based on phylogenetic analysis and is confirmed by the presence of conserved residues. Substrate-specific signature sequences (SSSSs) and specificity-determining positions (SDPs) predict the substrate specificity of each new aquaporin. Expression profiling in leaves, petioles and developing fruits reveals distinct patterns, even within the same (sub)class. Expression profiles range from leaf-specific expression over constitutive expression to fruit-specific expression. Both upregulation and downregulation during fruit ripening occur. Substrate specificity and expression profiles suggest that functional specialization exists among aquaporins belonging to a different but also to the same (sub)class.

Supplementing goat kids with coconut medium chain fatty acids in early life influences growth and rumen papillae development until 4 months after supplementation but effects on in vitro methane emissions and the rumen microbiota are transient.

The aim of this study was to investigate the methane (CH4) reducing potential of a combination of prenatal and/or postnatal treatment with coconut oil medium chain fatty acids (CO MCFA) in goat kids. The hypothesis is that influencing rumen function during early life has more chances for success than in the adult life, related to the resilience of the mature rumen microbiota. Forty-eight pregnant does were split into two experimental groups: treated does (D+) received 40 g/d of CO MCFA in a test compound feed, while control does (D-) received a control compound feed, during the last 3 wk of gestation. Twin kids from 10 does of each group were split up into a treated (K+) and nontreated (K-) group, resulting in four experimental groups: D+K+, D+K-, D-K+, and D-K-. The K+ kids received 1.8 mL/d of CO MCFA from birth until 2-wk postweaning (11 wk). Irrespective of treatment, the experimental rearing conditions resulted in absence of rumen protozoa at all sampling times, assessed by quantitative PCR (qPCR). In vitro incubations with rumen fluid at 4 wk old showed 82% lower CH4 production of inoculum from D+K+ kids compared to D-K- kids (P = 0.01). However, this was accompanied by lower total volatile fatty acids (tVFA) production (P = 0.006) and higher hydrogen accumulation (P = 0.008). QPCR targeting the mcrA and rrs genes confirmed a lower abundance of total methanogens (P < 0.02) and total eubacteria (P = 0.02) in D+K+ kids at 4 wk old. Methanogenic activity, as assessed by mcrA expression by RT-qPCR, was also lower in these kids. However, activity did not always reflect methanogen abundance. At 11 and 28 wk old, prenatal and postnatal effects on in vitro fermentation and rumen microbiota disappeared. Nevertheless, lower milk replacer intake in the first 4 wk resulted in reduced BW in K+ kids, persisting until 28 wk of age. Additionally, differences assigned to postnatal treatment were found in papillae density, width, and length in different areas of the rumen, recorded at 28 wk old. CONCLUSION: prenatal and postnatal supplementation with CO MCFA reduced in vitro CH4 emissions until 4 wk old by depressing methanogen abundance and activity but at the expense of rumen fermentation and eubacterial abundance. Unfortunately, daily gain of K+ kids was suppressed. Some rumen papillae characteristics differed at 28 wk old due to postnatal treatment which ended at 11 wk old, indicating rumen papillary development can be affected by the early-life nutritional circumstances.

Identification of Quantitative Trait Loci Controlling Root and Shoot Traits Associated with Drought Tolerance in a Lentil (Lens culinaris Medik.) Recombinant Inbred Line Population.

Drought is one of the major abiotic stresses limiting lentil productivity in rainfed production systems. Specific rooting patterns can be associated with drought avoidance mechanisms that can be used in lentil breeding programs. In all, 252 co-dominant and dominant markers were used for Quantitative Trait Loci (QTL) analysis on 132 lentil recombinant inbred lines based on greenhouse experiments for root and shoot traits during two seasons under progressive drought-stressed conditions. Eighteen QTLs controlling a total of 14 root and shoot traits were identified. A QTL-hotspot genomic region related to a number of root and shoot characteristics associated with drought tolerance such as dry root biomass, root surface area, lateral root number, dry shoot biomass and shoot length was identified. Interestingly, a QTL (QRSratioIX-2.30) related to root-shoot ratio, an important trait for drought avoidance, explaining the highest phenotypic variance of 27.6 and 28.9% for the two consecutive seasons, respectively, was detected. This QTL was closed to the co-dominant SNP marker TP6337 and also flanked by the two SNP TP518 and TP1280. An important QTL (QLRNIII-98.64) related to lateral root number was found close to TP3371 and flanked by TP5093 and TP6072 SNP markers. Also, a QTL (QSRLIV-61.63) associated with specific root length was identified close to TP1873 and flanked by F7XEM6b SRAP marker and TP1035 SNP marker. These two QTLs were detected in both seasons. Our results could be used for marker-assisted selection in lentil breeding programs targeting root and shoot characteristics conferring drought avoidance as an efficient alternative to slow and labor-intensive conventional breeding methods.

Change in Auxin and Cytokinin Levels Coincides with Altered Expression of Branching Genes during Axillary Bud Outgrowth in Chrysanthemum.

In the production and breeding of Chrysanthemum sp., shoot branching is an important quality aspect as the outgrowth of axillary buds determines the final plant shape. Bud outgrowth is mainly controlled by apical dominance and the crosstalk between the plant hormones auxin, cytokinin and strigolactone. In this work the hormonal and genetic regulation of axillary bud outgrowth was studied in two differently branching cut flower Chrysanthemum morifolium (Ramat) genotypes. C17 is a split-type which forms an inflorescence meristem after a certain vegetative period, while C18 remains vegetative under long day conditions. Plant growth of both genotypes was monitored during 5 subsequent weeks starting one week before flower initiation occurred in C17. Axillary bud outgrowth was measured weekly and samples of shoot apex, stem and axillary buds were taken during the first two weeks. We combined auxin and cytokinin measurements by UPLC-MS/MS with RT-qPCR expression analysis of genes involved in shoot branching regulation pathways in chrysanthemum. These included bud development genes (CmBRC1, CmDRM1, CmSTM, CmLsL), auxin pathway genes (CmPIN1, CmTIR3, CmTIR1, CmAXR1, CmAXR6, CmAXR2, CmIAA16, CmIAA12), cytokinin pathway genes (CmIPT3, CmHK3, CmRR1) and strigolactone genes (CmMAX1 and CmMAX2). Genotype C17 showed a release from apical dominance after floral transition coinciding with decreased auxin and increased cytokinin levels in the subapical axillary buds. As opposed to C17, C18 maintained strong apical dominance with vegetative growth throughout the experiment. Here high auxin levels and decreasing cytokinin levels in axillary buds and stem were measured. A differential expression of several branching genes accompanied the different hormonal change and bud outgrowth in C17 and C18. This was clear for the strigolactone biosynthesis gene CmMAX1, the transcription factor CmBRC1 and the dormancy associated gene CmDRM1, that all showed a decreased expression in C17 at floral transition and an increased expression in C18 with continuous vegetative growth. These results offer a case study for Chrysanthemum, showing an altered cytokinin to auxin balance and differential gene expression between vegetative growth with apical dominance and transition to generative growth with loss of apical dominance and axillary bud outgrowth. This suggests a conservation of several aspects of the hormonal and genetical regulation of bud outgrowth in Chrysanthemum. Furthermore, 15 previously uncharacterised genes in chrysanthemum, were described in this study. Of those genes involved in axillary bud outgrowth we identified CmDRM1, CmBRC1 and CmMAX1 as having an altered expression preceding axillary bud outgrowth, which could be useful as markers for bud activity.