Hypericum L. H2003-004-016 is a complex hybrid among Hypericum frondosum Michx., Hypericum galioides Lam., and Hypericum kalmianum L. and exhibits valuable ornamental characteristics, including compact habit, bluish green foliage, and showy flowers. Inducing polyploidy may further enhance the ornamental traits of this hybrid and provide new opportunities for hybridizing with other naturally occurring polyploid Hypericum sp. In this study, in vitro shoot regeneration and treatment of regenerative callus with the dinitroaniline herbicide oryzalin (3,5-dinitro-N4,N4-dipropylsufanilamide) were investigated as a means of inducing allopolyploidy. First, in vitro regeneration was optimized for callus and shoot induction by culture of leaf explants on medium supplemented with benzylamino purine (BA) or meta-topolin (mT) at 5, 10, or 15 μM in combination with indoleacetic acid (IAA) at 0, 1.25, 2.5, or 5 μM. Both BA and mT treatments successfully induced regenerative callus and shoots. Multiple regression analysis estimated maximum regenerative callus (94%) and shoot induction (18 shoots per explant) in medium supplemented with 5 μM BA and 3.75 μM IAA. In the second part of the study, exposure of regenerative callus to oryzalin at 0, 7.5, 15, 30, 60, or 90 μM for durations of 3, 6, or 9 d was investigated for polyploid induction. There was no survival for any of the calli in the 60- or 90-μM oryzalin treatments, but calli subjected to the other treatments exhibited some survival and polyploid induction. Duration had no effect on callus survival or ploidy level, but oryzalin concentration was a significant factor in both. The greatest percentage (44%) of polyploids was induced with 30 μM oryzalin. Spontaneous chromosome doubling was observed in 8% of control explants receiving no oryzalin treatment.
Elisabeth M. Meyer, Darren H. Touchell, and Thomas G. Ranney
Kelly M. Oates, Darren H. Touchell, and Thomas G. Ranney
Rudbeckia subtomentosa ‘Henry Eilers’ is an adaptable and popular garden plant; however, reduction in height and increased novelty in flower color would be desirable. The effect of gamma radiation dose on in vitro survival and development, and ex vitro fertility, phenology, and morphology of tetraploid Rudbeckia subtomentosa ‘Henry Eilers’ was investigated. In vitro embryogenic callus was treated with gamma radiation (0, 5, 10, 20, or 40 Gy). Rooted microshoots were established ex vitro and evaluated for morphology (plant height, number of flowers, diameter of the terminal flowers, diameter of the secondary flowers, number of stems, number of nodes, and internode length), date of first anthesis, winter survival, and pollen fertility on mature, second-year plants. Callus survival had no response to dose 2 months after treatment; however, microshoot number was significantly reduced with increasing dose. In vitro microshoot survival continued to decline at higher doses up to 4 months after treatment. Plant height, average stem height, number of flowers, flower diameter, percent winter survival, and pollen viability were all reduced with increasing radiation dose. Date of first anthesis was also delayed with increased radiation dose. Several off phenotypes were recorded including increased apical splitting of the ray florets in several plants. Gamma radiation was somewhat effective for reducing the height of R. subtomentosa ‘Henry Eilers,’ but flower morphology, flower number, and plant overwintering survival were often negatively affected with increasing radiation dose. Treating callus with low levels of gamma radiation (5–10 Gy) resulted in relatively high in vitro and field survival while inducing a range of other mutations that could be selected.
Kelly M. Oates, Thomas G. Ranney, and Darren H. Touchell
Rudbeckia spp. are adaptable and valuable ornamental wildflowers. Development of new varieties of Rudbeckia spp., with improved commercial characteristics, would be highly desirable. Interspecific hybridization and induced polyploidy may be avenues for improvement within the genus. The objective of this study was to evaluate fertility, morphology, phenology of flowering, and perennialness (overwintering survival) for lines of diploid and induced allotetraploids of R. subtomentosa × hirta and diploid and autotetraploids of R. subtomentosa ‘Henry Eilers’. Polyploid lines were developed and propagated in vitro and then grown ex vitro in a randomized complete block design with 12 replications. Compared with their diploid counterparts, autotetraploid lines of R. subtomentosa ‘Henry Eilers’ had similar internode lengths, plant heights, number of stems, flowering times (date at first anthesis), and fall and spring survival (100%); reduced number of inflorescences and male and female fertility; and increased inflorescence diameters. Compared with their diploid counterparts, allotetraploids of R. subtomentosa × hirta had similar internode lengths, reduced number of inflorescences, delayed flowering times, and increased pollen staining. Allotetraploids had limited male and female fertility compared with no detectable fertility in their diploid counterparts. Plant height and number of stems either decreased or showed no change with induced allotetraploidy. Spring survival of diploid hybrid genotypes ranged from 0% to 82% and was not improved in the allotetraploid hybrids. For a given genotype, some polyploidy lines varied significantly in certain morphological traits (e.g., plant height) indicating somaclonal variation may have developed in vitro or there were variable genomic or epigenetic changes associated with induced polyploidy.
Todd J. Rounsaville, Darren H. Touchell, and Thomas G. Ranney
Miscanthus sinensis Andersson is a popular ornamental grass and has additional potential as a bioenergy crop. In some regards, the ability of M. sinensis to withstand a broad range of climatic and cultural conditions is desirable, but its propensity to rapidly colonize open and disturbed environments has allowed it to naturalize and become weedy in some regions in the United States. Considering the value of this crop, the development and documentation of infertile clones would be desirable. Triploid plants were evaluated for male and female fertility using pollen viability staining and seed set and germination, respectively. Pollen viability staining, seed set, and seed germination from triploid plants were reduced compared with diploids but varied considerably within each cytotype. Overall, relative female fertility of individual triploids clones [(% seed set × % germination for triploid)/(% seed set × % germination for diploid control)] was reduced substantially and ranged from 49% to 0.7%. Additionally, the reproductive pathways of triploid plants were examined by evaluating the 2C genome sizes of progeny derived from open pollination. The limited progeny arising from open-pollinated triploids were predominantly aneuploids with 2C genome sizes intermediate between diploids and triploids. There was no clear evidence of apomixis, selfing, or triploid × triploid fertilization events observed among triploid parents. Formation of unreduced gametes was rare for both ploidy levels (≈1%). The considerable reduction in female fertility in some triploid clones combined with the limited production of primarily aneuploid progeny provides highly infertile alternatives to existing diploid cultivars.
Darren H. Touchell, Thomas G. Ranney, Dilip R. Panthee, Ronald J. Gehl, and Alexander Krings
Genetic diversity and cytogenetics of 31 accessions of Arundo L., collected from North America and South Asia, were characterized using 20 intersimple sequence repeat (ISSR) markers, flow cytometry, and cytology. In addition, field trials of 23 Arundo donax L. accessions were established in 2011 and harvested in 2012 to 2013 to assess annual biomass yields. Cluster analysis, based on Jaccard’s similarity coefficient method, clearly differentiated Arundo formasana Hack. from A. donax and a third unidentified Arundo taxon. Arundo donax further contained two subgroups representing North American (naturalized and cultivated) and South Asian collections. Within each A. donax subgroup, genetic distances were very low (0.03 for North America and 0.07 for South Asia). Principle coordinate analysis further supported distinct clusters. Relative genome sizes were determined using Pisum sativum L. as the reference genome and 6-diamidino-2-phenylindole (DAPI) fluorochrome. Chromosome numbers (2n), ploidy levels, and 2C relative genome sizes ranged from ≈62 to 105, near 12x to near 18x, and 2.78 to 4.13 pg, respectively, and were similar within each taxa/subgroup. While there was a low level of genetic variability among A. donax accession, dry biomass yields varied significantly ranging from 6.5 to 65 Mg·ha−1 per year for the third growing season.
Kelly M. Oates, Thomas G. Ranney, Darren H. Touchell, and Zenaida Viloria
J. Kevin Parris, Darren H. Touchell, Thomas G. Ranney, and Jeffrey Adelberg
In vitro growth responses of Magnolia ‘Ann’ to basal salt composition, cytokinins, and phenolic binding agents were investigated in a series of experiments to refine micropropagation protocols. Murashige and Skoog (MS), half-strength MS, Woody Plant Medium (WPM), Driver and Kuniyuki (DKW), and Blaydes basal salts in conjunction with 1 g·L−1 activated charcoal (AC) or 1 g·L−1 polyvinylpyrrolidone (PVP) were evaluated as multiplication media. Benzylaminopurine (BAP), meta-topolin (mT), or 6-(γ,γ-dimethylallylamino) purine (2iP) at 2, 4, or 8 μM was investigated to optimize the cytokinin concentration. Murashige and Skoog medium supplemented with 2 μM BAP with no phenolic binding agent was an optimal multiplication medium that yielded 3.2 ± 0.2 shoots with a mean length of 17.2 ± 1.8 mm over an 8-week period. For rooting, microshoots were cultured on half-strength MS media supplemented with 0, 5, 10, or 20 μM indolebutyric acid (IBA) with or without AC. Media containing AC produced elongated microshoots suitable for rooting and ex vitro establishment. Microshoots cultured on medium supplemented with AC also had higher in vitro rooting (16%) and higher ex vitro rooting (75%) compared with those without AC regardless of in vitro IBA concentration.
Todd J. Rounsaville, Darren H. Touchell, Thomas G. Ranney, and Frank A. Blazich
Mahonia ‘Soft Caress’ is a unique new cultivar exhibiting a compact form and delicate evergreen leaves. Protocols for micropropagation of M. ‘Soft Caress’ were developed to expedite multiplication and serve as a foundation for future work with other taxa of Mahonia Nutt. Combinations of sucrose at 30 or 45 g·L−1 in conjunction with Gamborg B5 (B5), Quoirin and Lepoivre (QL), and Murashige and Skoog (MS) basal media as well as other selected growth regulator treatments were evaluated as multiplication media. Rooting of microcuttings was conducted in vitro using combinations of indole-3-butyric acid (IBA) at 0, 2, 4, 8, or 16 μM under either light or dark. Quick dip treatments with aqueous solutions of the potassium (K) salt (K-salt) of IBA at 0, 5.2, 10.4, 20.7, or 41.4 μM were tested in a second experiment for ex vitro rooting. Media containing B5 basal salts and vitamins supplemented with sucrose at 30 g·L−1, 5 μM 6-benzylaminopurine, 5 μM kinetin, 0.5 μM indole-3-acetic acid, and 2.5 μM gibberellic acid yielded 2.80 ± 0.14 microshoots with a mean length of 14.76 ± 0.63 mm over a 6-week culture period and was an optimal multiplication media. Light treatment and IBA concentration had a significant effect on rooting percentages. Microcuttings treated with 8 μM IBA and maintained in the dark resulted in the best rooting (70%) and ex vitro establishment.
Whitney D. Phillips, Thomas G. Ranney, Darren H. Touchell, and Thomas A. Eaker
Flowering pears are popular landscape plants due to a combination of desirable traits including broad adaptability, pest resistance, and attractive ornamental features. However, in some areas, flowering pears readily reseed and naturalize. Considering the value and utility of these trees, the development of infertile cultivars would be desirable. Breeding of triploid plants is one of the approaches that has been successfully used to develop seedless cultivars of many crops. The objective of this study was to evaluate female fertility and reproductive pathways of triploid flowering pear hybrids. Female fertility was characterized by evaluating fruit set, seeds per fruit, seed germination, seedlings per flower, and percent relative fertility [(seedlings per flower for triploid/seedlings per flower for diploid control) × 100]. Flow cytometry was used to determine relative genome sizes and ploidy levels of female parents, seedlings, and seeds (both embryo and endosperm) and to make inferences regarding reproductive pathways. Mean holoploid genome sizes were confirmed for the diploid [1.25 ± 0.05 (se) pg] and triploid [1.88 ± 0.12 (se) pg] female parents. Relative female fertility was significantly reduced in triploids, but varied considerably among accessions and ranged from 0.0% to 33.6%. Of the 13 triploids used in this study, five accessions had a relative fertility of <2%. One accession had no measurable female fertility. Cytometric analysis of seeds and seedlings from triploid maternal parents showed that they were predominantly abnormal aneuploids, which typically results in seedlings with reduced fitness and fertility. Fertilization with unreduced gametes, apomixis, and pseudogamy were documented in triploid-derived embryos/offspring, but were relatively uncommon. The considerable reduction in female fertility of some triploid selections, coupled with the limited production of primarily aneuploid progeny, provides desirable options for new infertile flowering pears to prevent or reduce reseeding and naturalizing.
Cary J. Hebert, Darren H. Touchell, Thomas G. Ranney, and Anthony V. LeBude
Rhododendron L.‘Fragrantissimum Improved’ is an attractive cultivar with showy, fragrant flowers but has limited potential for breeding because it is a sterile wide hybrid. Protocols for in vitro regeneration and polyploid induction were developed for this cultivar as a means to potentially restore fertility and enhance ornamental traits. Combinations of thidiazuron (TDZ) at 0, 5, 10, 15, or 20 μM and 1-naphthaleneacetic acid (NAA) at 0, 2.5, 5, or 10 μM were used to induce shoot regeneration from leaves. Shoot regeneration was optimized (68% of leaf segments produced shoots) using 8.8 μM TDZ and 10 μM NAA. To induce polyploidy, regenerative callus was treated with 7.5, 15, 30, 60, or 90 μM of the mitotic inhibitor oryzalin for 1, 3, 5, 7, or 14 d in various combinations. Oryzalin significantly affected survival and shoot regenerative capacity. A percentage of homogenous, tetraploid shoots was recovered from treatments of 30 μM oryzalin for 1 (13%) or 3 (13%) days and 7.5 μM oryzalin for 7 (20%) or 14 (7%) days.