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.

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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.

Axillary bud outgrowth is regulated by both environmental cues and internal plant hormone signaling. Central to this regulation is the balance between auxins, cytokinins, and strigolactones. Auxins are transported basipetally and inhibit the axillary bud outgrowth indirectly by either restricting auxin export from the axillary buds to the stem (canalization model) or inducing strigolactone biosynthesis and limiting cytokinin levels (second messenger model). Both models have supporting evidence and are not mutually exclusive. In this study, we used a modified split-plate bioassay to apply different plant growth regulators to isolated stem segments of chrysanthemum and measure their effect on axillary bud growth. Results showed axillary bud outgrowth in the bioassay within 5 days after nodal stem excision. Treatments with apical auxin (IAA) inhibited bud outgrowth which was counteracted by treatments with basal cytokinins (TDZ, zeatin, 2-ip). Treatments with basal strigolactone (GR24) could inhibit axillary bud growth without an apical auxin treatment. GR24 inhibition of axillary buds could be counteracted with auxin transport inhibitors (TIBA and NPA). Treatments with sucrose in the medium resulted in stronger axillary bud growth, which could be inhibited with apical auxin treatment but not with basal strigolactone treatment. These observations provide support for both the canalization model and the second messenger model with, on the one hand, the influence of auxin transport on strigolactone inhibition of axillary buds and, on the other hand, the inhibition of axillary bud growth by strigolactone without an apical auxin source. The inability of GR24 to inhibit bud growth in a sucrose treatment raises an interesting question about the role of strigolactone and sucrose in axillary bud outgrowth and calls for further investigation

Helleborus species are members of the family of the Ranunculaceae. These popular perennials are all diploids (2n = 2x = 32). This study investigates polyploidy induction by different antimitotic agents. Colchicine, oryzalin and trifluralin were tested in vitro on shoots of Helleborus niger, H. orientalis and H. 9 nigercors. Furthermore the effect of the antimitotic agents on the viability and the multiplication rate of cultured plantlets were analyzed. Flow cytometry demonstrated that polyploidisation was genotype dependent: using H. niger, tetraploids were obtained using either oryzalin (3 lM) or trifluralin (3 or 10 lM), whereas for H. 9 nigercors only trifluralin (3 or 10 lM) induced polyploidisation. For H. orientalis neither treatment was effective to produce tetraploids or mixoploids. For these three species, colchicine (100 lM) was ineffective. The polyploidisation events in H. niger and H. 9 nigercors were confirmed by chromosome counts of mounted nuclei derived from root tips (2n = 4x = 64).

This study investigates the capacity of the antimitotic agents colchicine, oryzalin and trifluralin for inducing polyploidisation of Ranunculus asiaticus 'Alfa' in vitro shoots. Flow cytometry was used to evaluate the optimal concentration of each antimitotic agent for polyploidisation. Trifluralin at a concentration of 2 mu M resulted in the highest percentage of polyploidisation (27.5%), followed by a colchicine treatment of 200 mu M, which induced 23.3% of polyploids. For oryzalin the highest percentage was achieved using a concentration of 1 mu M. Different exposure periods were tested and turned out to be an important factor. The maximal exposure period tested (10 weeks) resulted in a significant increase in polyploidisation by oryzalin and trifluralin. In contrast, for colchicine (100 mu M) exposure times of either 16 or 24 h did not significantly influence polyploidisation. Additionally the effect of the antimitotic agents on the viability was analysed. For colchicine no significant effect on the survival rate was observed, for trifluralin only a concentration of 10 mu M affected viability whereas for oryzalin, concentration as well as exposure period were significant parameters. Flow cytometric data were confirmed by counting chromosomes in root tip cells

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