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Sandra M. Reed and Lisa W. Alexander

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Keri D. Jones and Sandra M. Reed

Previous attempts to use interspecific hybridization to combine flower color and cold hardiness in Hydrangea have not produced the desired results, with confirmed hybrids being weak, sterile or aneuploid. In all cases, H. macrophylla (Thumb.) Ser. was used as the source of flower color. This work investigates the use of H. involucrata Sieb. as an alternative source of flower color in Hydrangea interspecific hybridizations. Controlled reciprocal pollinations of H. involucrata with two cultivars of H. arborescens L. and three cultivars of H. paniculata Sieb. were made. Hybridity of progeny was verified using RAPD markers and confirmed with chromosome counts and morphological comparisons of hybrids and parents. Plants were obtained only when H. involucrata was used as the pollen parent. No hybrids between H. paniculata or H. arborescens `Annabelle' and H. involucrata were produced. Seven H. arborescens `Dardom' × H. involucrata progeny showed either a sum of the RAPD bands of both parents or banding patterns that matched those of H. involucrata. Leaf blade length and length/width ratio of the hybrid were intermediate to its parents. Chromosome number in the hybrid (2n = 34) was also intermediate between H. arborecens (2n = 38) and H. involucrata (2n = 30). One `Dardom' × H. involucrata plant flowered in 2005. While pollen staining indicated a very low level of fertility, we will continue to evaluate the possibility of using the hybrid for producing advanced filial or backcross progeny.

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Sandra M. Reed, Younghee Joung and Mark Roh

The genus Clethra contains many ornamental species, of which the most adaptable and cold hardy is C. alnifolia L. The objective of this study was to obtain hybrids between C. alnifolia and three other ornamental Clethra species, C. acuminata Michx., C. fargesii Franch., and C. pringlei S. Wats. Viable plants were obtained from reciprocal crosses between C. alnifolia and C. fargesii, and from crosses between C. alnifolia and the other two species when C. alnifolia was used as the maternal parent. Randomly amplified polymorphic DNA (RAPD) markers were used to verify hybridity and to compare hybrids to their parents. In all cases, the hybrids had more RAPD markers in common with C. alnifolia than with their other parent. Close clustering by neighbor-joining analysis of RAPD markers and the morphological resemblance of C. alnifolia × C. acuminata and C. fargesii × C. alnifolia plants to their paternal parent indicated that these plants were of hybrid origin. The C. alnifolia × C. pringlei plants resembled C. alnifolia in many respects, but they stayed green much later in the year than did C. alnifolia with leaves remaining on the plants throughout the winter. These foliage characteristics were presumed to reflect the contribution of the evergreen C. pringlei, and thus were regarded as evidence of hybridity.

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Sandra M. Reed and Margaret R. Pooler

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Sandra M. Reed and Timothy A. Rinehart

Genetic diversity studies using 26 simple-sequence repeat (SSR) markers were conducted with 36 cultivars, breeding lines, and wild-collected accessions of Hydrangea paniculata Sieb. The SSR markers were highly variable among the genotypes, producing a mean of 5.8 alleles per marker. Three cultivars (Boskoop, Compact Grandiflora, and Webb) were either identical to or sports of the popular cultivar Grandiflora. The name ‘Pee Wee’ appears to have been applied to two phenotypically different compact forms of H. paniculata, one of which seems to be a sport of ‘Tardiva’, whereas the other is likely derived from ‘Grandiflora’. No close genetic similarity was observed among several cultivars from a long-term Belgium breeding program, although many had one parent in common. Early-flowering genotypes clustered separately from genotypes that flower in midsummer, but close genetic relationships were not observed among early-flowering cultivars. Two genotypes from Taiwan were genetically similar but were distinctly different from the Japanese genotypes. These, along with the early-flowering genotypes and a new collection from Japan, may represent unexploited sources of germplasm for improvement of H. paniculata.

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Keri D. Jones, Sandra M. Reed and Timothy Rinehart

Dichroa febrifuga (Hydrangeaceae) is an evergreen shrub with metallic blue fruit that persists throughout winter. Phylogenetic studies have indicated that D. febrifuga is closely related to Hydrangea macrophylla. The objective of this study was to produce hybrids between these two ornamental species. Reciprocal crosses were made between D. febrifuga and both diploid and triploid cultivars of H. macrophylla. Two-thirds of the seed capsules produced from these hybridizations were allowed to mature on the plant, after which time seed were collected and sown in a greenhouse. Embryo rescue was used with the remaining seed capsules. Ovules were excised from ovaries about 9 weeks after pollination and plated on Gamborg's B-5 media with 2% sucrose. Both germination techniques resulted in vigorous seedlings. Hybridity was verified in plants obtained from ovule culture using simple sequence repeat (SSR) markers. Reciprocal hybrids were obtained using both diploid and triploid forms of H. macrophylla. This study confirms the close relationship between D. febrifuga and H. macrophylla and provides valuable material for future breeding efforts.

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Timothy A. Rinehart, Brian E. Scheffler and Sandra M. Reed

Using 14 codominant microsatellite markers that amplify loci across 14 different Hydrangea L. species, we analyzed gene diversity and genetic similarity within Hydrangea. Samples also included Dichroa Lour., Platycrater Sieb. and Zucc., and Schizophragma Sieb. and Zucc. genera to establish their relatedness to Hydrangea species since previous work suggests they may be closely related. Our results support the close affiliation between Macrophyllae E.M. McClint. and Petalanthe (Maxim.) Rehder subsections and their separation from the other Hydrangea species. Most of the Hydrangea species analyzed cluster within their designated sections and subsections; however, genetic distance between species within each subsection varied considerably. Our data suggest that morphological analyses which labeled H. serrata (Thunb.) Ser. as a subspecies of H. macrophylla (Thunb. Ex J.A. Murr.) Ser. are probably more accurate than recent genome size data suggesting H. macrophylla ssp. macrophylla (Thunb.) Ser. and H. macrophylla ssp. serrata (Thunb.) Makino are separate species. Gene diversity estimates indicate that 64.7% of the total diversity is due to differences between species and 49.7% of the overall variation is due to differences between subsections. Low diversity suggests a lack of gene flow between species and subsections and underscores the difficulty in making wide hybrids. Since only 35.3% of the genetic variation is common to all species, unique alleles were used to develop a molecular key for unambiguous species identification and interspecific hybrid verification. Genetic similarity estimates for all 85 samples suggests a roadmap for introgressing horticulturally important traits from different Hydrangea species.

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Sandra M. Reed, Keri D. Jones and Timothy A. Rinehart

The potential of producing an intergeneric hybrid between Dichroa febrifuga Lour. and Hydrangea macrophylla (Thunb.) Ser. was investigated. Reciprocal hybridizations were made between a D. febrifuga selection (GUIZ 48) and diploid (‘Veitchii’) and triploid (‘Kardinal’ and ‘Taube’) cultivars of H. macrophylla. Embryo rescue was employed for about one-third of the crosses that produced fruit, and the rest were allowed to mature on the plant and seed-collected and germinated. Reciprocal hybrids, which were verified with simple sequence repeat markers, were produced from both embryo rescue and seed germination and with both diploid and triploid H. macrophylla cultivars. Hybrids were intermediate in appearance between parents, but variability in leaf, inflorescence, and flower size and flower color existed among the hybrids. A somatic chromosome number of 2n = 6x = 108 was tentatively proposed for D. febrifuga GUIZ 48. Chromosome counts and flow-cytometric measurements of nuclear DNA content indicated that some of the hybrids may be aneuploids, but neither analysis was definitive. Although hybrids with H. macrophylla as the pistillate parent did not form pollen-producing anthers, D. febrifuga × H. macrophylla hybrids had normal-appearing anthers that produced abundant pollen. F2 and BC1 progeny were obtained using D. febrifuga × ‘Veitchii’ hybrids. This work documents the first step in an effort to combine desirable horticultural features from D. febrifuga and H. macrophylla.

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Keri D. Jones, Sandra M. Reed and Timothy A. Rinehart

Ploidy level was estimated in Hydrangea macrophylla (Thunb) Ser. using flow cytometry. For H. macrophylla ssp. macrophylla, 42 diploid and 19 triploid cultivars were identified. All 14 H. macrophylla ssp. serrata (Thunb.) Makino cultivars tested were diploids. Somatic chromosome counts confirmed the ploidy of three diploid (2n = 2x = 36) and three triploid (2n = 3x = 54) cultivars. Stomatal guard cell length and pollen diameter of H. macrophylla ssp. macrophylla diploid cultivars were smaller than those of triploid cultivars. However, because the range of measurements for the diploids overlapped that of the triploids, neither guard cell nor pollen measurements are recommended for determining ploidy of H. macrophylla cultivars. Fertility was estimated using pollen staining and controlled pollinations. Stainable pollen for triploid cultivars averaged 63% and ranged from 25% in ‘Masja’ to 85% in ‘Marechal Foch’. Viable seed was obtained when four triploid cultivars were used as pistillate or staminate parents in controlled pollinations to diploid H. macrophylla ssp. macrophylla cultivars. A bimodal distribution of pollen sizes, which is suggestive of unreduced gamete production, was observed in one cultivar; however, more detailed genetic and cytologic studies are needed to elucidate the mechanism behind triploid formation in H. macrophylla taxa.

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Sandra M. Reed, Gary R. Bachman and W. Edgar Davis