In vitro regeneration systems provide a powerful tool for manipulating ploidy to facilitate breeding and development of new crops. Polyploid induction can expand breeding opportunities, assist with the development of seedless triploid cultivars, enhance ornamental characteristics and environmental tolerances, increase biomass and restore fertility in wide hybrids. In vitro ploidy manipulation is commonly induced using antimitotic agents such as colchicine, oryzalin and trifluralin, while many other antimitotic agents have been relatively unexplored. Successful induction requires a synergistic pairing of efficient penetration of the antimitotic agent and may be dependent the length of exposure and concentrations of antimitotic agents, tissue types, and interactions with basal media and plant growth regulators. In vitro conditions vary among taxa and individual genera, species, and cultivars, often requiring unique treatments to maximize polyploid induction. In some taxa, the induction of polyploidy influences in vitro growth, development, and root formation. Here we provide an overview of mitotic inhibitors and their application for in vitro ploidy manipulation for plant breeding and crop improvement.

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Rudbeckia L. are valuable nursery crops that offer broad adaptability and exceptional ornamental merit. However, there is little information on interspecific and interploid crossability and ploidy levels of specific cultivars. The objectives of this study were to determine the ploidy levels and relative DNA contents (genome sizes) of selected species and cultivars, to evaluate self-compatibility and crossability among species and ploidy levels, and to explore reproductive pathways in triploid R. hirta L. with the goal of facilitating future breeding endeavors and development of new hybrids. Reciprocal interspecific crosses were performed between R. hirta cultivars and R. fulgida Ait., R. missouriensis Engelm. ex C.L. Boynton & Beadle, and R. subtomentosa Pursh. as well as reciprocal interploid crosses among four R. hirta cultivars. A combination of relative DNA content analysis and chromosome counts was used to test for hybridity and to determine ploidy levels for selected species, cultivars, and interploid R. hirta F1 hybrids. Of the specific clones tested, R. subtomentosa and R. missouriensis were diploid, R. fuligida varieties were tetraploid, and R. hirta include both diploid and tetraploid cultivars. Mean 1Cx DNA content varied over 320% among species. The interploid R. hirta crosses produced triploids as well as pentaploids and hexaploids. Seedlings from open-pollinated triploid R. hirta appeared, based on diverse phenotypes and DNA contents, to be aneuploids resulting from sexual fertilization, not apomixis. Of the 844 seedlings from interspecific F1 crosses, only one individual, R. subtomentosa ×R. hirta, had a DNA content intermediate between its parents and was confirmed as the only interspecific hybrid. Although most taxa had low self-fertility, seedlings (with genomic sizes similar to their maternal parent) resulted after interspecific crosspollination, indicating that pseudogamy is one reproductive pathway in Rudbeckia species.

Hydrangea macrophylla (Thunb.) Ser. and H. serrata (Thunb.) Ser. are popular and commercially important landscape and floriculture crops. Although both species are typically diploid, induced polyploids often exhibit horticulturally valuable traits. Procedures for inducing polyploidy vary by species and often have low or inconsistent efficacy. In this study, oryzalin and nitrotyrosine were investigated as in vitro mitotic inhibitors for inducing polyploidy in H. macrophylla ‘Robert’ and H. serrata ‘MAK20’. First, shoot apices of ‘MAK20’ were treated with 15 μm oryzalin for 0, 2, 4, 6, or 8 days, and the ploidy of shoots was determined after 8 weeks. A regression analysis showed that the proportion of polyploids (tetraploid plus mixoploid shoots) increased with the exposure duration. During a follow-up experiment, ‘MAK20’ and ‘Robert’ were treated with oryzalin (0 or 15 μm) and nitrotyrosine (0, 25, 50, and 100 µm for ‘MAK20’ and 0, 12.5, 25, 50, and 100 µm for ‘Robert’) in a factorial treatment arrangement. Oryzalin, nitrotyrosine, and their interaction influenced polyploid frequency for ‘Robert’, whereby the combination of oryzalin (15 μm) and nitrotyrosine (50 μm) resulted in the highest polyploid induction of 50%. Oryzalin influenced polyploid frequency for ‘MAK20’ ( = 30.4%), but not nitrotyrosine or the interaction between nitrotyrosine and oryzalin. Morphology and pollen germination of these autotetraploid ‘Robert’, ‘MAK20’, and previously developed autotetraploid H. macrophylla ‘David Ramsey’ plants were compared with their diploid counterparts 1 year after plants were moved ex vitro. Compared with diploids, tetraploid hydrangeas had larger leaves, thicker stems, lower leaf area/fresh weight ratios, and longer internodes. Although all tetraploids exhibited fewer inflorescences per plant, both H. macrophylla cultivars had larger inflorescence diameters and ‘David Ramsey’ had a greater number of showy florets (sterile florets with enlarged, decorative sepals) per inflorescence. Sepal colors were compared using International Commission on Illumination L*a*b* color space. Tetraploid ‘MAK20’ had lower L* values (darker sepals), and tetraploid ‘Robert’ and ‘MAK20’ both had higher a* values (redder sepals). Pollen germination rates were greatly reduced in all tetraploid lines, but they retained some viability. These results provide an effective protocol for in vitro polyploid induction of Hydrangea sp. and documented certain desirable traits associated with tetraploid phenotypes.

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