Althea (Hibiscus syriacus) is a shrub prized for its winterhardiness and colorful summer flowers. Altheas are tetraploids (2n = 4x = 80); however, breeders have developed hexaploids and octoploids. Previous studies report anatomical variation among polyploids, including stomata size. The purpose of this study was 4-fold. First, identify genome size and ploidy variation in cultivars via flow cytometry and chromosome counts. Second, create a ploidy series consisting of 4x, 5x, 6x, and 8x cytotypes. Third, investigate the ploidy series for variation in stomatal guard cell lengths, stomatal density, and copy number of fluorescent ribosomal DNA (rDNA) signals. Fourth, investigate segregation patterns of rDNA signals in a subset of pentaploid seedlings. Flow cytometry revealed most cultivars to be tetraploid with holoploid 2C genome sizes from 4.55 ± 0.02 to 4.78 ± 0.06 pg. Five taxa (‘Aphrodite’, ‘Pink Giant’, ‘Minerva’, Azurri Satin®, and Raspberry Smoothie™) were hexaploids (6.68 ± 0.13 to 7.05 ± 0.18 pg). Peppermint Smoothie™ was a cytochimera with tetraploid cells (4.61 ± 0.06 pg) and octoploid cells (8.98 ± 0.13 pg). To create pentaploids, reciprocal combinations were made between hexaploid ‘Pink Giant’ and tetraploid cultivars. To create octoploids, seedlings were treated with agar solutions containing 0.2% colchicine or 125 μM oryzalin. Guard cell lengths were significantly different among the four cytotypes: 4x (27.36 ± 0.04 μm), 5x (30.35 ± 1.28 μm), 6x (35.59 ± 0.63 μm), and 8x (40.48 ± 1.05 μm). Measurements of stomatal density revealed a precipitous decline in average density from the 4x cytotype (398.22 ± 15.43 stomata/mm2) to 5x cytotype (194.06 ± 38.69 stomata/mm2) but no significant difference among 5x, 6x, and 8x cytotypes. Fluorescent in situ hybridization (FISH) revealed an increase in 5S and 45S rDNA signals that scaled with ploidy: 4x (two 5S + four 45S), 6x (three 5S + six 45S), and 8x (four 5S + eight 45S). However, pentaploid (5x) seedlings exhibited random segregation of rDNA signals between the 4x and 6x cytotypes, including all six possible combinations (two 5S, three 5S) × (four 45S, five 45S, six 45S).

Liriope Lour. and Ophiopogon Ker Gawl., collectively known as liriopogons, represent important evergreen groundcovers grown throughout the world for their ornamental features and medicinal qualities. As a result of the diversity of desirable traits and evidence of wide hybridization, there is considerable potential for breeding and improvement of liriopogons. However, confusion over taxonomy and proper identification and lack of information on ploidy levels and cytogenetics of individual clones and cultivars have constrained breeding efforts. Objectives of this study were to validate the identification and nomenclature and determine genome sizes and ploidy levels for an extensive reference collection of species and cultivars of liriopogons. Identification was accomplished using existing keys, nomenclature was corrected, and numerous accessions were reassigned based on morphology. Genome sizes were determined by flow cytometry. Ploidy levels for each species were confirmed by traditional cytology. Results confirmed a basic chromosome number of x = 18 for liriopogons with aneuploidy, polyploidy, and cytochimeras found in some cases. The Liriope examined included diploids (L. graminifolia, L. longipedicellata, L. minor, and some of the L. platyphylla), tetraploids (L. muscari and the remaining L. platyphylla), and hexaploids (L. exiliflora and L. spicata). The Ophiopogon studied included diploids (O. intermedius, O. jaburan, O. planiscapus, and O. umbraticola) and a tetraploid/hypotetraploid species (O. japonicus). Monoploid (1Cx) genome sizes varied by genus and species with 1Cx values ranging from 4.27 pg in L. exiliflora to 8.15 pg in O. jaburan. These results clarify nomenclature and taxonomy and provide specific information on genome sizes and ploidy levels of cultivated liriopogons. This information and associated reference collection will aid future taxonomic revisions and enhance efforts to develop new cultivars of liriopogons.

Protocols were developed for micropropagation and induction of autopolyploids in a fastigiate cultivar of Norway maple (A. platanoides L. ‘Crimson Sentry’). Murashige and Skoog (MS) medium, woody plant medium (WPM), and Quoirin and Lepoivre medium were supplemented with 2 μM 6-benzylaminopurine (BA), meta-Topolin, 6-(γ,γ-dimethylallylamino) purine, kinetin, or thidiazuron to evaluate microshoot proliferation. Murashige and Skoog medium with 2 μM BA yielded the most microshoots (3.2) and longest microshoots (30.6 mm) per subsample after 5 weeks. The influence of BA concentration on proliferation was evaluated at 0, 2, 4, 8, or 16 μM. Optimal multiplication rate was achieved at 2 or 4 μM BA producing approximately 2.8 microcuttings per subsample after 5 weeks. To induce in vitro rooting, half-strength WPM was supplemented with 0, 5, 10, 20, 40, or 80 μM indole-3-butyric acid (IBA). Optimal in vitro rooting (70%), number of roots (2.5), and root length (15 mm) per subsample were achieved with 10 μM IBA after 8 weeks. To induce polyploidy, microcuttings were pretreated for 7 days on MS medium with 4 μM BA alone or combined with 1 μM IBA, indole-3-acetic acid (IAA), or 1-naphthaleneacetic acid prior to treatment in liquid MS medium containing 15 μM oryzalin for 3 days. Homogenous tetraploids were only obtained from shoots pretreated with IAA. This research provides optimized protocols for micropropagation and autopolyploid induction of A. platanoides ‘Crimson Sentry’ and demonstrates the development of tetraploid lines for use in future improvement programs.

Chromosome numbers are an important botanical character for multiple fields of plant sciences, from plant breeding and genetics to systematics and taxonomy. Accurate chromosome counts in root tips of woody plants are often limited by their small, friable roots with numerous, small chromosomes. Current hydrolysis and enzyme digestion techniques require handling of roots before the root squash. However, optimum chromosome spread occurs when the cell walls have degraded past the point of easy handling. Here, we present a new enzyme digestion protocol that is fast, efficient, and flexible. This protocol reduces handling of the roots allowing for long-duration enzyme digestion. Digestions are performed on a microscope slide, eliminating the need for handling digested cells with forceps or pipettes. To illustrate the flexibility of this method across woody plant taxa, we performed chromosome counts on five angiosperms and one gymnosperm. Ploidy levels included diploids, triploids, and tetraploids with chromosome numbers ranging from 2n = 16 to 2n = 80. The range of holoploid 2C genome sizes spanned 1.54–24.71 pg. This protocol will provide a useful technique for plant cytologists working with taxa that exhibit a wide range of genome size and ploidy levels.

Lilacs (Syringa sp.) are a group of ornamental trees and shrubs in the Oleaceae composed of 22–30 species from two centers of diversity: the highlands of East Asia and the Balkan-Carpathian region of Europe. There are six series within the genus Syringa: Pubescentes, Villosae, Ligustrae, Ligustrina, Pinnatifoliae, and Syringa. Intraspecific and interspecific hybridization are proven methods for cultivar development. However, reports of interseries hybridization are rare and limited to crosses among taxa in series Syringa and Pinnatifoliae. Although hundreds of lilac cultivars have been introduced, fertility and cross-compatibility have yet to be formally investigated. Over 3 years, a cross-compatibility study was performed using cultivars and species of shrub-form lilacs in series Syringa, Pubescentes, and Villosae. A total of 114 combinations were performed at an average of 243 ± 27 flowers pollinated per combination. For each combination, we recorded the number of inflorescences and flowers pollinated as well as number of capsules, seed, seedlings germinated, and albino seedlings. Fruit and seed were produced from interseries crosses, but no seedlings were recovered. A total of 2177 viable seedlings were recovered from interspecific and intraspecific combinations in series Syringa, Pubescentes, and Villosae. Albino progeny were produced only from crosses with Syringa pubescens ssp. patula ‘Miss Kim’. In vitro germination was attempted on 161 seed from interseries crosses, resulting in three germinations from S. pubescens Bloomerang® x Syringa vulgaris ‘Ludwig Spaeth’. None survived, yet cotyledons produced callus for future efforts to induce embryogenic shoots. This study is a comprehensive investigation of lilac hybridization, and the knowledge gained will aid future efforts in lilac cultivar development.