Flower longevity is an important character in many ornamental crops. The processes of pollination and fertilization can cause senescence of the petals through the action of ethylene or its precursors. Preventing the production of pollen and therefore pollination could delay the senescence of petals. We tested whether male-sterility would increase flower longevity in petunia. The gene consisted of a stamen-specific promoter isolated from a Lycopersicon esculentum gene driving the expression of a barnase. Barnase is a RNase that is cytotoxic. The gene was introduced into `Lavender Storm' and `Purple Wave' petunia by Agrobacterium- mediated gene transfer. Five independent transgenic lines of both cultivars were regenerated, rooted, and grown in a greenhouse. All lines showed complete male-sterility as measured by the lack of detectable pollen. Two transgenic lines and a non-transformed control of each cultivar were propagated vegetatively and the flower longevity of each genotype was determined in a greenhouse experiment. There were two treatments: no pollination or pollination with cross-compatible pollen. All sterile genotypes that were not pollinated had increased flower longevity relative to pollinated sterile flowers or either treatment of male fertile (non-transformed) genotypes. These results indicate an application for sterility in the production of petunia flowers with increased longevity. Male and female sterility may be applicable in other ornamental crops where pollination or fertilization is a trigger to petal senescence.
Alan G. Smith*, Nicole Gardner and Elizabeth Zimmermann
Alan G. Smith and Kenneth J. McNeil
The sporophytic tissue of the anther and, in particular, the tapetum, a cell layer surrounding the pollen sac, is know to be essential for the production of pollen. The isolation and characterization of the gene 92B from tomato that encodes an extracellular glycine rich protein (GRP) has been used to further elucidate the role of the tapetum in pollen development. RNA from the 92B gene accumulates exclusively in the tapetum. Polyclonal antibodies raised against the 92B GRP detect four proteins in stamens with microspores beginning meiosis. In pollen extracts, the antibodies detect a single protein. Expression of the tomato 92B gene in transgenic tobacco indicates that the four protein products are derived from only the 92B gene. The 92B GRP is localized to the tapetum, the callose wall of microspore mother cells, the exine (outer wall) of mature pollen, and orbicules. Orbicules are globular bodies derived from tapetal material that form on the tapetum wall and line the exterior of the pollen sac. Expression of 92B antisense RNA resulted in a significant decrease of 92B RNA and protein levels in transgenic tomatoes. This reduction was correlated with a decrease in pollen germination and an abnormal exine morphology. The function of the 92B protein in pollen development and function will be discussed.
Alan G. Smith and Elizabeth S. Zimmermann
Euonymus alata is an attractive landscape plant that has been reported to be an invasive species. Genetic modification through transformation is a method of reducing its invasiveness by producing sterile cultivars having limited or no seed production. A critical step in Agrobacterium-mediated gene transfer is the production of adventitious shoots. E. alata internodes and leaves from in vitro cultures were tested for adventitious shoot production on 16 plant growth regulator combinations: four levels of 6-benzylamino purine (BA) and three auxin treatments [0.5 or 0.25 mg·L-1 indole-3-butyric acid and 0.1 mg·L-1 naphthaleneacetic acid (NAA)], as well as no auxin. The optimal BA levels were found to be 0.5 or 1.0 mg·L-1 for maximizing the number of explants forming shoots and for producing the greatest number of shoots per explant. Culturing on NAA gave the greatest number of shoots per explant with both 0.5 and 1.0 mg·L-1 BA. Shoot production from internode segments was markedly superior to leaves. An initial dark treatment of 10 days did not influence shoot production. Using 1.0 mg BA with 0.1 mg·L-1 NAA, E. alata internodes were transformed with A. tumefaciens EHA105 carrying Kanamycin resistance and β-glucuronidase genes. Transformed shoots were selected on 30 mg·L-1 Kanamycin. Of the 36 shoots produced, 16 were confirmed to be transformed by β-glucuronidase histochemistry. Treatment with rooting powder containing indole-3-butyric acid did not aid rooting of shoots, but after 3 months in soil in high humidity, 21 of 24 E. alata shoots from tissue culture were rooted and acclimated.
Alan G. Smith*, Nicole Gardner and Tracy A. Melberg
As residential lot sizes decrease, there is an increased demand for new, small-statured landscape plants to fit into the smaller lots. One promising method to create smaller plants is by introducing a dwarfing gene into a plant of interest. A dwarfing gene that has been identified is the rolC gene from Agrobacterium rhizogenes. Expression of rolC in plants has been shown to cause decreased height and internode length, increased branching, and modified leaf size in a several species. Although the effects of the rolC gene have been well characterized for many plant species, most research has concerned the native promoter or the CaMV 35S promoter. Less research has been done with additional promoters or comparing the results from different promoters. In this study we examined the effects of three separate gene constructs, all containing rolC driven under either the 35S promoter, the light inducible rbcs promoter, or the native rolC promoter in tobacco. Plants transformed with these constructs ranged widely for height and other phenotypic traits. Representative plants were crossed back to wild-type tobacco. Plants from this next generation, six with the 35S promoter, six with the rbcs promoter and four with the native rolC promoter, were measured for traits such as height, days to flower, number of branches and internode length. RolC RNA expression levels were also measured in roots, stems, and leaves to determine correlations between rolC expression level in specific tissues and the observed phenotype. Information about these relationships can be used to provide insight into the use of rolC in ornamental plants and the potential to modify its phenotypic effects by controlling expression level.
Esther E. McGinnis, Alan G. Smith and Mary H. Meyer
Pennsylvania sedge (Carex pensylvanica) is an upland forest sedge with restoration and horticultural potential as a low-maintenance groundcover for dry shade. For large landscape and restoration plantings, seed or achenes in this case are much preferred due to lower labor and material costs. However, pennsylvania sedge typically produces few achenes in its native habitat. As a first step in improving achene production, this research evaluated the effect of vernalization and photoperiod on floral initiation and development. We conclude that this sedge is an obligate short-day plant that does not require vernalization for flowering. Plants flowered when exposed to daylengths of 6 to 12 hours. Flowering was completely inhibited with 14-hour photoperiods. Pennsylvania sedge was florally determined after 4 weeks of 8-hour photoperiods. Inflorescence quantity and normal floral development varied by clone and by weeks of exposure to 8-hour photoperiods. For two of the clones, the largest number of normal monoecious inflorescences was produced with 8 to 10 weeks of 8-hour photoperiods while the other two clones only required 6 to 8 weeks of exposure to inductive photoperiods. Therefore, it is important to evaluate observable variation between clones when attempting to propagate pennsylvania sedge.
Troy D. Carson, Donald B. White and Alan G. Smith
Poa annua L. has long been a cultivated weed on golf courses. However, the recent development of improved cultivars of creeping bluegrass (Poa annua reptans Hausskn.) has generated an increasing interest in selection and breeding of this species. Inter-simple sequence repeat (ISSR) PCR is a relatively new method for genotype identification and measuring genetic diversity and was employed in this study for differentiating among creeping bluegrass genotypes. The objectives of this study were to test ISSR primers for production of polymorphic fragments and ascertain the applicability of ISSR PCR to distinguish closely related genotypes. Eight primers produced fragments, of which 77.3% were polymorphic, and primers UBC849, UBC850, and UMN001 produced over 75% of the total polymorphic fragments. These three primers had sufficient resolution to distinguish all but two of the diploid creeping bluegrass accessions. This method was a simple, fast, and relatively inexpensive method to produce useful DNA fragments in creeping bluegrass. It is a robust method for detecting polymorphic loci that can be used in the study of genetic relatedness, heritability, or linkage to important traits, development of linkage maps, and marker-assisted breeding.
Nicole Gardner, Tracy Melberg, Manju George and Alan G. Smith
Expression of the rolC gene in plants has been shown to cause pleiotropic effects, including decreased height. The effects of differential rolC gene expression on plant height, leaf color, root growth, leaf size, corolla length, and stem diameter were determined. Differential expression of rolC in Nicotiana tabacum L. `Samsun' plants was achieved using the 35S promoter, the light-inducible rbcS promoter, or the native rolC promoter. Sixteen plants from the T1 generation—six with the 35S promoter, six with the rbcS promoter, and four with the native rolC promoter—and non-transformed controls were measured for height, internode length, branch number, bud size, corolla length and diameter, root growth, and the number of days to flowering. Steady state mRNA levels of rolC were measured in roots, stems, and leaves to assess relationships between rolC expression level in specific tissues and phenotypes. Plants expressing rolC showed a wide range of phenotypes, with the largest changes in plants expressing rolC using the 35S promoter, which also had the highest rolC mRNA levels. Plants expressing rolC with the rolC or rbcS promoter had significant changes for many measured traits, despite rolC mRNA levels that were not significantly different from non-transformed controls. In general, as rolC mRNA levels increased, so did the severity of the rolC phenotype observed. Three plants, A4, A7, and B9, had unique combinations of traits that did not follow this general trend. Transformation with rolC can be useful in ornamental crops where smaller cultivars are desired.
Andrzej K. Noyszewski, Neil O. Anderson, Alan G. Smith, Andrzej Kilian, Diana Dalbotten, Emi Ito, Anne Timm and Holly Pellerin
In cases where invasive species are presumed to be strictly exotic, the discovery that the species is also native can be disconcerting for researchers and land managers responsible for eradicating an exotic invasive. Such is the case with reed canarygrass (Phalaris arundinacea), for which decades of misinformation led to the call for nationwide control of this species in the United States. However, native populations were first reported by LaVoie and then later confirmed by Casler with molecular analyses. This, coupled with the discovery by Anderson that this species has been used in weavings by Native Americans for centuries, also made the native forms of interest for protection. Identifying the native status of historic, herbarium specimens via molecular analyses is of great interest to determine localities of native populations for confirmation with extant specimens. Genetic-based methods describing DNA polymorphism of reed canary grass are not well developed. The goal of the presented research is to assess the utility of genomic DNA obtained from historic (herbaria) and extant (fresh) tissue of reed canarygrass and the application of using Diversity Arrays Technology sequencing low density for genetic population studies.