The genus Clematis contains many well-known large flowered cultivars, as well as lesser-known nonvining species. Intersimple sequence repeat (ISSR) primers were used to fingerprint 32 vining cultivars and five nonvining species (C. fruiticosa, C. integrifolia, C. heracleifolia, C. hexapetala, and C. recta) for use in assessing genetic relationships and cultivar identification. Four ISSR primers yielded a total of 44 bands in the vining accessions, of which 36 (86%) were polymorphic. The average polymorphism levels were 83% for the cultivars and 94% for the nonvining species. All 32 vining cultivars were distinguished with the use of two ISSR primers, and the five nonvining Clematis species were differentiated with three ISSR primers. A similarity matrix of the cultivars showed low similarity levels between the samples, with an average similarity of 0.28. A UPGMA-derived dendrogram showed no strong groupings among any of the samples. Two cultivars with known parentage, Clematis viticella L. `Betty Corning' and `Sylvia Denny', grouped with one reported parent but not the other, suggesting they are more similar to one parent. `Multi-blue', a sport reportedly arising from `The President' did not segregate near `The President'.
Nicole Gardner and Stan C. Hokanson
Alan Smith, Nicole Gardner, and Elizabeth S. Zimmermann
Female sterility is desirable in horticultural crops for many reasons, including decreasing invasiveness and weediness, reducing nuisance fruit production, promoting vegetative growth, and increasing flower longevity and number. This study tested a method of creating female sterility through genetic transformation of plants with a gene that ablates tissue necessary for female fertility and reproduction. A gene construct was created containing barnase, a cytotoxic RNAse, expressed with a transmitting tract specific promoter from the tobacco gene sP41. The sP41gene encodes a (1,3)-β-glucanase in the transmitting tract of the pistil of mature tobacco flowers. The construct also expressed barstar, a barnase inhibitor, driven by the CaMV 35S promoter to protect other plant tissues from unexpected barnase expression. Seed set data taken after controlled pollinations showed tobacco plants transformed with this construct had greatly reduced fertility in young flowers and female sterility in mature flowers relative to nontransformed controls. Light microscopy showed ablation of the transmitting tract tissue in transformed plants. The expression of barnase with a transmitting tract specific promoter is an effective way to reduce or eliminate female fertility. Due to the conservation of (1,3)-β-glucanase activity in the styles of other plant species, this construct has potential for producing female-sterile cultivars of other horticultural crops.
Alan G. Smith*, Nicole Gardner, and Elizabeth Zimmermann
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.
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.
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.