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Luping Qu and Mark P. Widrlechner

Prunella vulgaris (Lamiaceae), commonly known as selfheal, is a perennial herb with a long history of use in traditional medicine. Recent studies have found that P. vulgaris possesses anti-inflammatory, antiviral, and antibacterial properties, and it is likely that this will lead to increased commercial demand for this species. To date, research publications on P. vulgaris cultivation and genetics are scarce. Using accessions originally collected from different geographical regions, we investigated the breeding system of this species by observing variation in floral morphology, time of pollen release, and selfed-seed set in bagged flowers and isolated plants. Two types of floral morphology, one with exerted styles, extending past open corollas when viewed from above, and the other with shorter, inserted styles, were found among 30 accessions. Two accessions originally collected from Asia uniformly displayed exerted styles, and 27 accessions had inserted styles. One accession from Oregon displayed variation in this trait among individual plants. Microscopic observation of seven accessions, including ones with both exerted and inserted styles, revealed that they all release pollen to some degree before the flowers open. Using bagged flowers, we found that selfed-seed set varied widely among eight accessions, ranging from 6% to 94%. However, bagging may underestimate seed set for some accessions. The two accessions with the lowest rates when using bagged flowers increased in seed set by 350% and 158%, respectively, when we evaluated single, unbagged plants in isolation cages. The accession with 6% selfed-seed set when bagged also had exerted styles. These findings suggest that mating systems in P. vulgaris may be in the process of evolutionary change and that understanding breeding-system variation should be useful in developing efficient seed-regeneration protocols and breeding and selection strategies for this species.

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Luping Qu and James F. Hancock

A tetraploid blueberry population resulting from a cross of US 75 {a tetraploid hybrid of Fla 4B [a selection of Vaccinium darrowi Camp (2n = 2x = 24) × `Bluecrop' [(V. corymbosum L. (2n = 4x = 48)]} × `Bluetta' (4x) was used to generate a genetic linkage map of US 75 by randomly amplified polymorphic DNA (RAPD) analysis. One hundred and forty markers unique for Fla 4B that segregated 1:1 in the population were mapped into 29 linkage groups that cover a total genetic distance of 1288.2 cM, with a range of 1.6 to 33.9 cM between adjacent markers. The map is essentially of V. darrowi because US 75 was produced via a 2n gamete from Fla 4B and only unique markers for Fla 4B were used. Therefore, all the chromosomes of V. darrowi could be represented in the map.

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Luping Qu and J.F. Hancock

RAPD markers were used to determine the level of heterozygosity transmitted via 2n gametes from V. darrowi cv. Florida 4b (Fla 4B) to interspecific hybrids with tetraploid V. corymbosum cv. Bluecrop. The tetraploid hybrid US 75 was found to contain 70.6% of Fla 4B's heterozygosity, a value consistent with a first division restitution (FDR) mode of 2n gamete production. Crossovers during 2n gamete formation were evidenced by the absence of 16 dominant alleles of Fla 4B in US 75, and direct tests of segregation in a diploid population involving Fla 4B. RAPD markers that were present in both Fla 4B and US 75 were used to determine the mode of inheritance in a segregating population of US 75 × V. corymbosum cv. Bluetta. More than 30 homozygous pairs of alleles were located that segregated in a 5:1 ratio, indicating US 75 undergoes tetrasomic inheritance.

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Luping Qu, James Polashock and Nicholi Vorsa

Putative transgenic cranberry plants have been achieved via Agrobacterium-mediated transformation. Leaf explants were transformed with a supervirulent Agrobacterium tumefaciens strain EHA 105, harboring the binary vector P35SGUSint and nptII selectable marker genes. Inoculation of precultured explants (≈10 days on regeneration medium) coupled with sonicasion improved transformation efficiency significantly. Adventitious shoots were directly regenerated from explants. Putative transformed shoots were identified by being kanamycin-resistant and GUS-positive. Stable GUS gene expression (turning blue) could be detected within 1 h of incubation at 37 °C. Confirmation of transformation by molecular analysis is in progress. Eight putative transgenic cranberry plants were obtained. All appeared morphologically normal. This appears to be the first success in achieving cranberry transformed plants by Agrobacterium-mediated method. Optimizing the transformation system is ongoing.

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Luping Qu, James Polashock and Nicholi Vorsa

We have established a very efficient cranberry regeneration (shoot organogenesis) system from leaf explants using a basal medium consisting of Anderson's salts and Murashige and Skoog (MS) organics supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) and N6-(-??-dimethyallylamino) purine) (2ip). Characteristics examined include combinations of varying levels of three plant growth regulators (TDZ, 2ip, and naphthaleneacetic acid (NAA), explant orientation (adaxial or abaxial side in contact with the media), and leaf position relative to the distal end of the shoot. Genotypes (`Early Black', `Pilgrim', `Stevens', `Ben Lear', and US#35) differed significantly in regeneration capacity, and there were no genotype by treatment interaction effects. Regeneration occurred on more than 95% of the explants with `Early Black' and `Pilgrim' producing as many as 100 shoot tips per explant with one particular treatment. Emerging adventitious shoots were always observed on the adaxial side of the leaves regardless of explant orientation. However, regeneration was much greater when the adaxial side was in contact with the media. Regeneration efficiency was not significantly affected by leaf position (10 leaves). Elongation of shoot tips began about 2 weeks after the regenerating explants were transferred to the basal medium without hormones and continued for several months. Elongated shoot cuttings rooted readily.

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Luping Qu, James Polashock and Nicholi Vorsa

A very efficient adventitious regeneration (shoot organogenesis) system for cranberry (Vaccinium macrocarpon Ait.) leaves was developed. A basal medium consisting of Anderson's rhododendron salts and Murashige and Skoog's (MS) organics, supplemented with 10.0 μm thidiazuron (TDZ) and 5.0 μm 2ip, was effective for adventitious regeneration from leaves for the five cranberry cultivars tested: `Early Black', `Pilgrim', `Stevens', `Ben Lear', and `No. 35'. Parameters examined included: 1) varying combinations of three plant growth regulators (TDZ, 2ip, and NAA); 2) explant orientation (adaxial vs. abaxial side in contact with the medium); and 3) leaf position relative to the apical meristem from the source plant. Cultivars varied in regeneration frequency, but cultivar × growth regulator interaction was nonsignificant. With optimal treatment conditions, regeneration occurred on more than 95% of the explants, with `Early Black' and `Pilgrim' producing as many as 100 shoot meristems per explant. At all concentrations tested, NAA (as low as 0.1 μm) increased callus formation and significantly reduced regeneration. Emerging adventitious shoots were always observed on the adaxial side of the leaves regardless of explant orientation on the medium. Regeneration was much greater when the abaxial side was in contact with the medium, and was not related to leaf position on the source plants. Elongation of adventitious shoots began ≈2 weeks after transfer to the basal medium without growth regulators. Cuttings of elongated shoots rooted 100% both in vitro in the basal medium and ex vitro in shredded sphagnum moss. The high regeneration efficiency achieved by using this system will be very useful in the application of techniques, such as Agrobacterium- and particle bombardment-mediated transformation. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ); N6-(γ-γ-dimethyallylamino) purine (2ip); α-naphthaleneacetic acid (NAA).

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Luping Qu, Xiping Wang, Eatherley Hood, Meihua Wang and Richard Scalzo

Chromosome karyotypes of the most commonly cultivated and medicinally used Echinacea taxa, E. angustifolia DC. var. angustifolia and E. purpurea (L.) Moench., were analyzed. The chromosomes of both taxa are medium in length, ranging from 4.12 to 5.83 μm in E. angustifolia var. angustifolia and 3.99 to 6.08 μm in E. purpurea. No abrupt length changes in the chromosomes were noted. The karyotypes of the two species are generally similar, but a distinguishable feature exists in one pair of chromosomes. The centromere of chromosome pair 10 is subterminally located in E. purpurea, but terminally located in E. angustifolia var. angustifolia, which can be readily recognized in mitotic metaphase cell plates. This finding may provide useful information for Echinacea evolutionary, genetic, and breeding studies.

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Luping Qu, Xiping Wang, Jinghua Yang, Eatherley Hood and Richard Scalzo

Seeds from five lots each of Echinacea angustifolia DC and E. pallida (Nutt.) Nutt. were germinated in a growth chamber in light (40 μmol·m-2·s-1) or darkness at 25 °C for 16 to 20 days after soaking in 1 mm ethephon or water for 10 minutes or moist stratification at 4 - 6 °C for 2 weeks. Either light or ethephon promoted seed germination of E. angustifolia and E. pallida compared with darkness in nine of ten lots. Ethephon in the dark had similar or greater germination percentages than water with light. Ethephon with light improved germination in three of ten lots compared with ethephon in the dark. The effect of cold, moist stratification compared with darkness varied by seed lot. Five lots of E. purpurea (L.) Moench were tested, however, no treatment differences were measured. The finding that ethethon promoted E. angustifolia and E. pallida seed germination in darkness could be useful in the cultivation of these two species. Chemical name used: 2-chloroethylphosphonic acid (ethephon).

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Luping Qu, Ying Chen, Xiping Wang, Richard Scalzo and Jeanine M. Davis

We investigated patterns of variation in alkamides and cichoric acid accumulation in the roots and aboveground parts of Echinacea purpurea (L.) Moench. These phytochemicals were extracted from fresh plant parts with 60% ethanol and quantified by high performance liquid chromatography (HPLC) analysis. Concentrations of alkamides and cichoric acid were measured on a dry-weight basis (mg·g–1). For total alkamides, concentrations among individual plants varied from 5.02 to 27.67 (mean = 14.4%) in roots, from 0.62 to 3.42 (mean = 1.54) in nearly matured seed heads (NMSH), and 0.22 to 5.25 (mean = 0.77) in young tops (about ½ flower heads, ¼ leaves, and ¼ stems). For cichoric acid, concentrations among individual plants varied from 2.65 to 37.52 (mean = 8.95), from 2.03 to 31.58 (mean = 10.9), and from 4.79 to 38.55 (mean = 18.88) in the roots, the NMSH, and the tops, respectively. Dodeca-2E, 4E, 8Z, 10E-tetraenoic acid isobutylamide and dodeca-2E, 4E, 8Z, 10Z-tetraenoic acid isobutylamide (alkamides 8/9) accounted for only 9.5% of the total alkamides in roots, but comprised 87.9% in the NMSH, and 76.6% in the young tops. Correlations of concentrations of alkamides or cichoric acid between those of roots and those of the NMSH were not statistically significant, and either within the roots, the NMSH, and the young tops. However, a significant negative correlation was observed between the concentration of cichoric acid in the roots and in young tops, and a significant positive correlation was observed between total alkamide concentration in the roots and cichoric acid concentration in the young tops. These results may be useful in the genetic improvement of E. purpurea for medicinal use.

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Luping Qu, Xiping Wang, Ying Chen, Richard Scalzo, Mark P. Widrlechner, Jeanine M. Davis and James F. Hancock

Seed germination patterns were studied in Echinacea purpurea (L.) Moench grouped by seed source, one group of seven lots from commercially cultivated populations and a second group of nine lots regenerated from ex situ conserved wild populations. Germination tests were conducted in a growth chamber in light (40 μmol·m–2·s–1) or darkness at 25 °C for 20 days after soaking the seeds in water for 10 minutes. Except for two seed lots from wild populations, better germination was observed for commercially cultivated populations in light (90% mean among seed lots, ranging from 82% to 95%) and in darkness (88% mean among seed lots, ranging from 82% to 97%) than for wild populations in light (56% mean among seed lots, ranging from 9% to 92%) or in darkness (37% mean among seed lots, ranging from 4% to 78%). No germination difference was measured between treatments in light and darkness in the commercially cultivated populations, but significant differences were noted for treatments among wild populations. These results suggest that repeated cycles of sowing seeds during cultivation without treatments for dormancy release resulted in reduced seed dormancy in E. purpurea.