Tom Eeckhaut

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.

Progress in plant protoplast research

Abstract In this review we focus on recent progress in protoplast regeneration, symmetric and asymmetric hybridization and novel technology developments. Regeneration of new species and improved culture techniques opened new horizons for practical breeding in a number of crops. The importance of protoplast sources and embedding systems is discussed. The study of reactive oxygen species effects and DNA (de)condensation, along with thorough phytohormone monitoring, are in our opinion the most promising research topics in the further strive for rationalization of protoplast regeneration. Following, fusion and fragmentation progress is summarized. Genomic, transcriptomic and proteomic studies have led to better insights in fundamental processes such as cell wall formation, cell development and chromosome rearrangements in fusion products, whether or not obtained after irradiation. Advanced molecular screening methods of both genome and cytoplasmome facilitate efficient screening of both symmetric and asymmetric fusion products. We expect that emerging technologies as GISH, high resolution melting and next generation sequencing will pay major contributions to our insights of genome creation and stabilization, mainly after asymmetric hybridization. Finally, we demonstrate agricultural valorization of somatic hybridization through enumerating recent introgression of diverse traits in a number of commercial crops.

Exploitation of flow cytometry for plant breeding

In this review, the different applications of flow cytometry in plant breeding are highlighted. Four main breeding related purposes can be distinguished for flow cytometry: (i) Characterisation of available plant material, including screening of possible parent plants for breeding programs as well as evaluation of population biodiversity; (ii) Offspring screening after interspecific, interploidy or aberrant crosses; (iii) Ploidy level determination after haploidization and polyploidization treatments and (iv) Particle sorting, that allows separation of plant cells based on morphological or fluorescent characteristics. An overview and discussion of these various applications indicates that flow cytometry is a relatively quick, cheap and reliable tool for many breeding related objectives.

Somatic polyploid petals: regeneration offers new roads for breeding Belgian pot azaleas

Bud sporting, the consequence of sudden variations in gene expression of somatic cells, leads to the occurrence of phenotypically altered shoots in many vegetatively propagated plant species. In ornamentals, such as azalea (Rhododendron simsii), flower colour bud sports are appreciated as a valuable additional source of variation, and it is evident that both genetic and epigenetic mechanisms are involved in the creation of the whole colour range. Flowers exhibiting two-coloured petals, i.e. the margin is coloured differently than the central zone, characterize one of the sport types, namely picotees. This particular sporting type contributes to the enlargement of the available variation not only because of its coloration pattern but interestingly displays somatic polyploidy in its petals. The differently coloured petal margin is tetraploid while the rest of the corolla, as well as the entire plant are diploid. Aiming at introducing polyploidy in the completely diploid breeding collection, in vitro regeneration of tetraploid petal marginal tissue was performed. This led to the production of the first induced tetraploid Belgian azalea. The markedly increased robustness of the flowers as well as their polyploid status is exceptionally interesting for breeding programs.

In vitro polyploidy induction in Rhododendron simsii hybrids.

Three in vitro protocols for the induction of tetraploid Rhododendron simsii hybrids were evaluated. The most successful technique was a daily application of mitotic poison on the apical meristem of seedlings. However, this technique was rather labour intensive. Direct sowing on media enriched with these compounds required less work but was less efficient. Mitotic inhibitors, when added to multiplication media, induced ploidy chimeras of existing genotypes.

Micropropagation of Rhododendron

Methods for in vitro initiation and multiplication and general culture practices of Rhododendron are presented. Also acclimatization procedures are described. Protocols for callus, shoot, and root induction are described. Several protocols for breeding applications are highlighted in more detail.

Karyotype analysis and visualization of 45S rRNA genes using fluorescence in situ hybridization in aroids (Araceae).

Abstract Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species Anthurium andraeanum Linden ex André, 1877, Monstera deliciosa Liebmann, 1849, Philodendron scandens Koch & Sello, 1853, Spathiphyllum wallisii Regel, 1877, Syngonium auritum (Linnaeus, 1759) Schott, 1829 and Zantedeschia elliottiana (Knight, 1890) Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendron scandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllum wallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthurium andraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschia elliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies.

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.

OPTIMISATION OF SOMATIC HYBRIDISATION IN CICHORIUM SPECIES

Improving the inulin production in the root of industrial chicory by conventional breeding has reached its limit. Therefore, we aimed to increase genetic variability in industrial chicory (Cichorium intybus) to enhance its inulin production. To this end, we executed symmetric protoplast fusions between industrial chicory on the one hand and endive or a wild type Cichorium intybus foliosum on the other hand. In order to produce plantlets, efficient protocols needed to be established for protoplast fusion and regeneration, and for the screening of the putative hybrids. We created a regeneration protocol by which we can produce plantlets out of protoplasts using low melting point agarose beads surrounded by liquid medium. Two weeks after protoplast isolation, 60% of the initial incubated protoplasts showed microcolony development in the beads. After transfer to solid medium, containing 0.5 mg L-1 IAA and 0.5 mg L-1 BAP, shoots were induced on the developing calli. Three months after protoplast isolation, the first plants were recovered. Protoplasts from different genotypes of C. intybus sativum and C. intybus foliosum could be regenerated. Symmetric protoplast fusion was performed using PEG-induced, chemical fusion. Industrial chicory (C. intybus sativum), endive (C. endivia crispum) and wild chicory (C. intybus foliosum) were used as fusion partners. Up to 30% of the regenerants after fusion between industrial chicory and endive were hybrids. The fusion of industrial chicory with wild chicory produced on average 4% hybrid regenerants. Microsatellite markers confirmed the hybridity of the putative fusion products. These findings enable us to produce, fuse and regenerate protoplasts of different Cichorium species in a rapid and efficient way. Moreover, the methods for symmetric protoplast fusion and regeneration presented in this study, can directly contribute to further research on asymmetric protoplast fusion experiments between these three species. This way, CMS could possibly be introduced by combining the nucleus of industrial chicory with the cytoplasm of other Cichorium types.