Emmy Dhooghe

Mitotic chromosome doubling of plant tissues in vitro

In vitro chromosome doubling can be induced by several antimitotic agents. The most commonly used are colchicine, oryzalin and trifluralin. The process of induced chromosome doubling in vitro consists of a typical succession of sub-processes, including an induction phase and a confirmation protocol to measure the rate of success. The induction step depends on a large number of variables: media, antimitotic agents, explant types, exposure times and concentrations. Flow cytometry is the pre-eminent method for evaluation of the induced polyploidization. However, alternative confirmation methods, such as chromosome counts and morphological observations, are also used. Since polyploidization has many consequences for plant growth and development, chromosome doubling has been intensively studied over the years and has found its way to several applications in plant breeding. This review gives an overview of the common methods of chromosome doubling in vitro, the history of the technique, and progress made over the years. The applications of chromosome doubling in a broader context are also discussed.

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.

Branching gene expression during chrysanthemum axillary bud outgrowth regulated by strigolactone and auxin transport

Shoot branching is essential in ornamental chrysanthemum production and determines final plant shape and quality. Auxin is associated with apical dominance to indirectly inhibit bud outgrowth. Two non-mutually exclusive models exist for indirect auxin inhibition. Basipetal auxin transport inhibits axillary bud outgrowth by limiting auxin export from buds to stem (canalization model) or by increasing strigolactone levels (second messenger model). Here we analyzed bud outgrowth in treatments with auxin (IAA), strigolactone (GR24) and auxin transport inhibitor (NPA) using a split-plate bioassay with isolated chrysanthemum stem segments. Besides measuring bud length, dividing cell percentage was measured with flow cytometry and RT-qPCR was used to monitor expression levels of genes involved in auxin transport (CmPIN1) and signaling (CmAXR2), bud dormancy (CmBRC1, CmDRM1) and strigolactone biosynthesis (CmMAX1, CmMAX3). Treatments over a 5-day period showed bud outgrowth in the control and inhibition with IAA and IAA + GR24. Bud outgrowth in the control coincided with high dividing cell percentage, decreased expression of CmBRC1 and CmDRM1 and increased CmPIN1 expression. Inhibition by IAA and IAA + GR24 coincided with low dividing cell percentage and unchanged or increased expressions of CmBRC1, CmDRM1 and CmPIN1. Treatment with GR24 showed restricted bud outgrowth that was counteracted by NPA. This restricted bud outgrowth was still concomitant with a high dividing cell percentage and coincided with decreased expression of dormancy genes. These results indicate incomplete inhibition of bud outgrowth by GR24 treatment and suggest involvement of auxin transport in the mechanism of bud inhibition by strigolactones, supporting the canalization model.

Ploidy Breeding in Ornamentals

Ploidy alterations are frequently used in ornamental plant breeding to induce novel variation, to overcome crossing barriers, or to create homogenous lines. Here we review three in vitro ploidy breeding tools with proven practical potential: mitotic polyploidization using mitosis arresting chemicals, meiotic polyploidization through the application of 2n gametes, and haploidization through either male or female gamete regeneration or sexual hybridization techniques. For each tool, we present the state of the art, summarize recent progress, and report on the relative success of different approaches. We summarize the technical procedures of those techniques and their advantages, drawbacks, and repercussions for plant breeding. For mitotic polyploidization, we compare the effects of mitotic inhibitors through meristem treatment, short exposure, and chronic exposure. For unreduced gamete formation via chemicals, we describe N2O exposure and temperature treatment. For haploid induction, we compare possibilities of parthenogenesis, microspore embryogenesis, and gynogenesis/androgenesis. To conclude, we present the perspectives for further research in all of these fields. In the future we expect progress in elucidating molecular backgrounds of 2n gamete formation and their interaction with the environment and the implementation of CENH3 modification as an alternative for classical haploidization techniques. Although our goal is not to provide detailed protocols, we have compiled a basic manual for ploidy breeding. This text gives technical insights into the different induction methods and correlates those to consequences for use in practical breeding.

Production and Characterization of Intergeneric Hybrids between Anemone coronaria and Ranunculus asiaticus

Anemone coronaria L. and Ranunculus asiaticus L. are common cut flowers which belong to the Ranunculaceae. Between these species a high degree of variation can be found in leaves, flower morphology and flower colour. Therefore intergeneric crossings between these species might result in new interesting hybrids. Crosses between these genera were performed in the greenhouse and pre- and post-zygotic barriers were examined. Two to five weeks after pollination, immature achenes were harvested and rescued in vitro. The obtained F1 generation was investigated on morphological, molecular and cytogenetic level. The F1 plants had very similar flowers to the mother plants and although the limited molecular paternal contribution, AFLP analyses confirmed the hybrid character of the F1 plants. Chromosome counts showed that the F1 plants were mixoploids, composed of cells with different chromosome numbers.