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Alan T. Whittemore and Zheng-Lian Xia

( Goldblatt and Johnson, 1979 ; Oginuma et al., 1990 ). Polyploidy is rare, and it is known from only two species. U. americana is known to include tetraploids with 2 n = 56 ( Karrfalt and Karnosky, 1975 ) as well as diploids ( Whittemore and Olsen, 2011

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Alan W. Meerow, Timothy K. Broschat, and Michael E. Kane

An amaryllis breeding program using diploid species not represented in commercial tetraploid cultivars has been underway since 1988. Objectives are to develop evergreen cultivars with attractive foliage and fragrant flowers of novel form and coloration. Five crosses with Hippeastrum papilio as a parent were evaluated at first flowering in the spring of 1990. The F-1's showed significant variation, suggestive of high heterozygosity within the parental genomes. Several natural tetraploids were identified among the progeny. Superior selections were made, and sib- or intercrosses accomplished. We estimate that a minimum of 50% genes from H. papilio will need to be maintained to guarantee evergreen foliage in the progeny. Superior F-1's have also been crossed with fragrant, trumpet-flowered primary hybrids, and new primary F-1's are being generated with H. papilio and these species or their hybrids, as well as with H. reticulatum var. striatifolium. A percentage of these germinated seedlings have been treated with colchicine to induce polyploidy. The best F-1 selections are also being micropropagated, and induction of polyploidy will be attempted in a percentage of the subcultures.

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Bruce L. Dunn and Jon T. Lindstrom

Ploidy level and fertility status are often the two biggest barriers a breeder must overcome when trying to incorporate novel characteristics among related taxa. This study was aimed at developing an efficient chromosome doubling method for Buddleja L., commonly known as butterfly bush, with the goal of equalizing the ploidy level and restoring the fertility of a diploid (2n=38) F1 interspecific hybrid that has a unique orange color but happens to be sterile. This method would ease the crossing of the hybrid to the tetraploid (2n=76) B. davidii Franch. cultivars commonly found in the industry. An antimitotic treatment of oryzalin was tested on 02-25-142 (B. madagascarensis Lam. × B. crispa Benth.) in vitro using nodal sections. A factorial of varying concentrations [3, 5, and 7 μM (micromolar)] by different exposure times (1, 2, and 3 day) plus controls was set up. Oryzalin appeared to be an efficient agent for chromosome doubling in Buddleja. Significant differences in the number of polyploids were not seen between chemical concentrations and exposure times. However, higher chemical concentrations and exposure times did have a significant effect on the number of nodes that survived tissue culture. Increased leaf size and color, stem thickness, shortened internode length, and upright growth habit were all good early phenotypic indicators of polyploidy induction as later confirmed by flow cytometry. Significant increases in pollen viability accompanied chromosome doubling as crosses between 02-25-142 × B. davidii cultivars produced viable seedlings.

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Supriyo Basak, Guangyan Wang, Xudong Sun, and Yongping Yang

Brassica rapa var. rapa (turnip) is considered a main source of food for the inhabitants of the Qinghai-Tibetan Plateau (QTP) and its adjacent highlands when other crops are scarce. The QTP ranges from lat. 25.59°N to 39.49°N and from long. 73.29°E to 104.40°E, whereas the Yunnan Plateau ranges from lat. 20.00°N to 29.16°N and from long. 96.00°E to 110.19°E. A comparison between the turnip landraces of two different plateau environments can provide a mechanistic insight into plant adaptation in highlands. The aim of this investigation was to understand the patterns in variation in genome size (GS) between the turnip landraces of two plateau environments. We used a well-established protocol to count chromosome number and performed propidium iodide flow cytometry to measure GS. No polyploidy was detected among the turnip landraces tested, and 15.5% variation in GS was observed between the landraces. No consistent pattern pertaining to GS variation emerged after the environmental variables were considered. Thus, we propose that such pattern may reflect the indirect effect of selection, random process, genetic drift, or some other factors on GS through interaction of life-form and phenotypic traits.

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Q. Liu, S. Salih, J. Ingersoll, R. Meng, L. Owens, and F. Hammerschlag

Transgenic `Royal Gala' apple (Malus × domestica Borkh.) shoots were obtained by Agrobacterium-mediated gene transfer using the plasmid binary vector pGV-osm-AC with a T-DNA encoding a chimeric gene consisting of a secretory sequence from barley-amylase joined to the modified cecropin MB39 coding sequence. Shoots were placed under the control of a wound-inducible, osmotin promoter from tobacco. The integration of the cecropin MB39 gene into apple was confirmed by Southern blot analysis. The transformation efficiency was 1.5% when internodes from etiolated shoots were used as explants and 2% when leaf explants were used. Both non- and transgenic tetraploid plants were produced by treatment of leaf explants with colchicine at 25 mg·L-1, and polyploidy was confirmed by flow cytometry. Of the diploid transgenics, three of seven were significantly more resistant to Erwinia amylovora than the non-transgenic `Royal Gala' control. Also, in one instance, a tetraploid transgenic was significantly more resistant than the diploid shoot from which it was derived.

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Laura L. Benson, Warren F. Lamboy, and Richard H. Zimmerman

The U.S. National Plant Germplasm System (NPGS) currently holds 36 separate accessions of the `Yichang' clone of Malus hupehensis (Pamp.) Rehd. The `Yichang' clone originally entered the United States in 1908 as seed collected for the Arnold Arboretum by E.H. Wilson near Yichang, Hubei Province, China. The original description of M. hupehensis omits fruit characters, and botanists frequently augment these omissions with descriptions of the `Yichang' clone. Apomixis occurs in Malus, including M. hupehensis, and is strongly associated with elevated ploidy levels. Simple sequence repeats (SSRs) were used to characterize 65 accessions of M. hupehensis. To check for polyploidy, a set of M. hupehensis accessions was evaluated with flow cytometry. The simple sequence repeat phenotypes and ploidy information revealed the `Yichang' clone under various accession names in arboreta. It was neither known nor suspected that the U.S. National Plant Germplasm System held many duplicate accessions of the `Yichang' clone prior to their molecular characterization. Germplasm conservation decisions for Malus species can benefit from an increased knowledge of the genetic variation or lack thereof in naturalized populations and ex situ collections.

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Young A Choi, Ryutaro Tao, Keizo Yonemori, and Akira Sugiura

5S ribosomal DNA (rDNA) was visualized on the somatic metaphase chromosome of persimmon (Diospyros kaki) and ten wild Diospyros species by fluorescent in situ hybridization (FISH). The digoxigenin (DIG)-labeled 5S rDNA probe was hybridized onto the chromosomes and visualized by incubation with anti-DIG-fluorescein isothiocyanate (FITC). Strong signals of 5S rDNA probe were observed on several chromosomes of Diospyros species tested. Furthermore, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of Diospyros species tested, suggesting that 5S and 45S rDNA sites can be used as chromosome markers in Diospyros. The number of 5S rDNA sites varied with the Diospyros species. More 5S rDNA sites were observed in four diploid species native to Southern Africa than in three Asian diploid species. The former had four or six 5S rDNA sites while the latter had two. Three Asian polyploidy species had four to eight 5S rDNA sites. Among the Asian species, the number of 5S rDNA sites seemed to increase according to ploidy level of species. These features of 5S rDNA sites were very similar to those of 45S rDNA sites in Diospyros. Phylogenetic relationship between D. kaki and wild species tested are discussed based on the number and chromosomal distribution of 5S and 45S rDNA.

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Kittipat Ukoskit and Paul G. Thompson

Low-density randomly amplified polymorphic DNA (RAPD) markers of sweetpotato [Ipomoea batatus (L.) Lam.; 2n = 6x = 90] were constructed from 76 pseudotestcross progenies obtained from `Vardaman' × `Regal'. Of 460 primers, 84 generating 196 well-resolved repeatable markers were selected for genetic analysis. `Vardaman' and `Regal' testcross progenies were analyzed for segregation and linkages of RAPD markers. Type of polyploidy, autopolyploidy, or allopolyploidy is uncertain in sweetpotato and was examined in this study using the ratio of nonsimplex to simplex RAPD markers and the ratio of simplex RAPD marker pairs linked in repulsion to coupling. Both measures indicated autopolyploidy. Low-density RAPD linkage maps of `Vardaman' and `Regal' were constructed from simplex RAPD marker linkage analysis. Duplex and triplex markers were then mapped manually into the simplex marker map. Homologous linkage groups were identified using nonsimplex RAPD markers and three homologous groups were found in each of the parent maps. Use of nonsimplex markers increased mapping efficiency. The `Vardaman' map had a predicted coverage of 10.5% at a 25-cM interval of the genome size of 5024 cM. In `Regal', genome coverage was estimated to be 5.6% at a 25-cM interval of the genome size of 6560 cM. Therefore, average chromosome length was ≈56 to 73 cM.

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Ryan N. Contreras and Thomas G. Ranney

Wide hybridization can potentially lead to the combination of diverse traits, but these hybrids are often sterile as is the case with the inter-subgeneric hybrid Rhododendron `Fragrant Affinity'. Induction of polyploidy can restore chromosome homology and fertility in wide hybrids. In this study we successfully developed an allopolyploid form of R. `Fragrant Affinity' using oryzalin as a mitotic inhibitor and chromosome doubling agent. Approximate genome size (2C), determined using flow cytometry, was 1.6 pg for the diploid and 3.2 pg for the allotetraploid. Pollen viability, determined by staining and germination tests, was 4% and 0%, respectively for the diploid and 68% and 45%, respectively for the allotetraploid. No seeds were produced when the diploid R. `Fragrant Affinity' was crossed with pollen from viable diploid and tetraploid parents. The allotetraploid produced viable seeds and seedlings when crossed with viable pollen from either diploid or tetraploid parents, including self pollination, demonstrating restored fertility. Additional crosses were successfully completed using the allotetraploid as part of an ongoing breeding program to develop new fragrant, cold hardy, evergreen rhododendron.

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H. Yamane, R. Tao, A. Sugiura, N. Hauck, and A. Iezzoni

Most fruit tree species of Prunus exhibit gametophytic self-incompatibility, which is controlled by a single locus with multiple alleles (S-alleles). One interesting aspect of gametophytic self-incompatibility is that it commonly “breaks down” as a result of polyploidy, resulting in self-compatible individuals. This phenomenon is exhibited in the diploid sweet cherry (P. avium) and the tetraploid sour cherry (P. cerasus), in which most cultivars are self-compatible. Recently, S-gene products in pistil of Prunus species were shown to be S-RNases. As sour cherry is one Prunus species, it is likely to possess S-alleles encoding pistil S-RNases. To confirm this, we surveyed stylar extracts of 11 sour cherry cultivars, including six self-compatible and five self-incompatible cultivars, by 2D-PAGE. As expected, all 11 cultivars tested yielded glycoprotein spots similar to S-RNases of other Prunus species in terms of Mr, immunological characteristics, and N-terminal sequences. A cDNA clone encoding one of these glycoproteins was cloned from the cDNA library constructed from styles with stigmas of a self-compatible cultivar, `Erdi Botermo'. Deduced amino acid sequence from the cDNA clone contained two active sites of T2/S type RNases and five conserved regions of rosaceous S-RNases. In order to determine the inheritance of self-incompatibility and S-allele diversity in sour cherry, we conducted genomic DNA blot analysis for sour cherry germplasm collections and mapping populations in MSU using the cDNA as a probe. To date, it appears as if self-compatibility in sour cherry is not simply controlled by a self-fertile allele as demonstrated in other Prunus species.