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Richard H. Ozminkowski Jr. and Pablo Jourdan

Brassica napus (genome aacc), a natural allotetraploid derived from hybridization between B. oleracea L. (genome cc) and B. rapa L. (genome aa), was resynthesized by somatic and sexual hybridization. Seventy-two interspecific somatic (R0) hybrids and 27 sexual (F1) hybrids were produced from the same parent plants. R0 and F1 hybrids displayed morphology that was intermediate to the species parents, but B. rapa characteristics tended to predominate. R0 hybrids with nuclear DNA content equivalent to natural B. napus were uniform for nuclear-encoded traits, whereas allotetraploid F1 hybrids were variable for traits such as morphology, flower color, and seed production. Chloroplast restriction fragment length polymorphisms (RFLPs) showed unequal segregation in the R0 population favoring the chloroplasts of B. rapa; two of the 58 R0 hybrids tested had only the B. oleracea marker and 10 contained markers of both parents. Mitochondrial RFLPs showed a similar bias among the 56 R0 hybrids tested; only four plants showed B. oleracea markers exclusively, and the remaining plants were evenly distributed between having only B. rapa markers or having combinations from both species. In contrast, sexual hybrids displayed only maternal organelle markers.

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Robert H. Bors and J. Alan Sullivan

Interspecific crosses with Fragaria moschata (6x) have been hampered by ploidy level differences, poor seed set, and extremely poor seed germination. Modification of pollination practices, embryo rescue, and use of several genotypes has allowed over 80 synthetic tetraploids to be created from 14 cross combinations. Germplasm for the experiment consisted of eight selections of F. moschata (6x), two of F. nubicola (2x), and two of F. viridis (2x). Both 2x × 6x and 6x × 2x crosses were performed. Initially, negligible seed set occurred on F. nubicola and F. viridis when multiple flowers per truss were pollinated. When only one cross was performed per truss, with other flowers removed, seed set was greatly enhanced. F. moschata was much more tolerant of multiple crosses per truss. The crossing combination of F. moschata × F. nubicola gave the worst seed production. Other species combinations were capable of producing good seed set with noticeable differences between individual selections. When achenes were halved, only 1% appeared normal, 2% were underdeveloped or shrunken, the remainder were empty. Many of the malformed and most of the normal embryos germinated using the cut achene method. Achenes were surface-sterilized, cut in half, and placed on MS media with activated charcoal (3g·L–1), sucrose (30g·L–1), and no hormones. Germination occurred only from achenes from fully ripened fruit. Viable hybrids were obtained from 2x × 6x as well as 6x × 2x crosses. Fragaria viridis–F. moschata hybrids closely resembled F. moschata while F. nubicola–F. moschata hybrids were more intermediate in leaf morphology.

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Mark P. Widrlechner

In 1991, the USDA–ARS North Central Regional Plant Introduction Station made available for distribution 129 accessions of germplasm representing 31 genera of herbaceous ornamentals. This number increased to 329 accessions of 42 genera by 1995. During 1991–95, more than 500 seed packets were distributed to fulfill requests for these plants received from a diverse array of public and private researchers. An analysis of this demand together with expert advice from Crop Germplasm Committees and technical considerations, such as ease of culture and seed production, can help set priorities to plan germplasm regeneration to meet future demand. A recent analysis of demand at U.S. National Plant Germplasm System active sites indicated that demand ranging between 0.23 and 0.97 distributions per available accession per year was typical. Of the 42 ornamental genera analyzed in this study, 9 were demanded more frequently than was typical, 10 were demanded less frequently, with the remainder in the typical range. In order of increasing frequency, the nine genera with the highest distribution rates were Verbena, Gypsophila, Echinacea, Lapeirousia, Delphinium, Cerastium, Baptisia, Lilium, and Tanacetum. Six of these genera are represented only by a single available accession. Notably, Echinacea and Tanacetum are of research interest both as ornamentals and as medicinal/industrial crops. This poster gives a brief overview of the economic value of these genera, display the results of the demand analysis, discuss the results relative to recommendations from Crop Germplasm Committees and requestors, and consider how demand can shape management plans for the acquisition and regeneration of ornamental germplasm.

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Roberto Nuñez-Elisea and Jonathan H. Crane

Carambola (Averrhoa carambola L.) is native to the humid tropics of southeastern Asia, where it bears fruit year-round. In south Florida, winter conditions (strong winds and night temperatures below 15 °C) repress growth and flowering of the main commercial cultivar, Arkin, and fruit is produced from July to February. Off-season fruit would reach premium prices. We have previously demonstrated that selective pruning stimulates flowering of carambola at any time of the year. However, flowers produced during cool, windy weather have consistently failed to set fruit. This study was conducted in 1994–1995 to determine whether protected cultivation would help obtain off-season fruit. Four-year-old `Arkin' trees growing in 80-L containers were placed in a glasshouse or outdoors and pruned in November or December to force flowering during December–January. Glasshouse night temperatures during the winter were above 20 °C. All trees flowered in response to pruning. Outdoor trees produced less than one fruit per tree in late March to late April. Glasshouse trees produced 2.3 to 6.1 fruit per tree, 2 to 3 weeks earlier than trees outdoors. In the glasshouse, more than 98% of fruit were seedless, whereas all fruit produced outdoors were seeded. Production of seedless fruit indoors was achieved in the absence of insect pollinators, and yields were low compared to those of outdoor trees during the summer (at least 25 fruit per tree). We speculate that, under protected cultivation, the use of synthetic bioregulators during anthesis and insect pollinators may help increase production of off-season seedless and seeded fruit, respectively.

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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.

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Orville C. Baldos, Joseph DeFrank, and Glenn S. Sakamoto

Tanglehead (Heteropogon contortus) is a native Hawaiian grass that has been used in restoration and has potential for expanded re-vegetation use. Although interest and demand for tanglehead re-vegetation has increased, the supply of tanglehead seeds has remained limited as a result of a lack of seed production protocols addressing seed dormancy. Smoke water from burning vegetation may provide an economical and practical seed treatment because aerosol smoke has been reported to stimulate tanglehead seed germination. Dose rate and side-by-side comparison studies were conducted to evaluate the germination stimulation efficacy of food-grade liquid smoke, xylose smoke-infused water, tanglehead smoke-infused water, karrikinolide (KAR1), and cyanide (i.e., mandelonitrile and potassium cyanide). Optimum smoke water dilutions were 1% v/v for food-grade liquid smoke and undiluted for xylose smoke-infused water and tanglehead smoke-infused water. KAR1 was not stimulatory at concentrations between 0.0067 and 66.7 μm. Potassium cyanide stimulated tanglehead seed germination at concentrations between 50 to 500 μm. Germination was promoted to even greater levels with the cyanohydrin, mandelonitrile, indicating a role for benzaldehyde (a byproduct of mandelonitrile decomposition) in stimulating tanglehead seed germination. Benzaldehyde was confirmed to be stimulatory at concentrations between 50 to 100 μm. The presence of cyanide at stimulatory levels was confirmed in tanglehead smoke-infused water (i.e., ≈100 μm), but not in food-grade liquid smoke or xylose smoke-infused water. Germination with non-cyanide-containing smoke waters indicates the presence of other compounds in smoke that can stimulate tanglehead germination. In the side-by-side comparison study, food-grade liquid smoke (1% v/v) and undiluted tanglehead smoke-infused water provided consistent germination stimulation comparable to 500 μm potassium cyanide. Undiluted xylose smoke-infused water did not provide significant germination stimulation in the comparison studies. This may be the result of differences in seed batch sensitivity to the germination stimulant, seed storage duration as well as subtle differences in the preparation of xylose smoke-infused water.

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Sullivan Lynch, Rachel K. Johnston, Ron O. Determann, Jennifer M. Cruse-Sanders, and Gerald S. Pullman

Symphyotrichum georgianum (Asteraceae), commonly known as Georgia aster, is a candidate for listing under the Federal Endangered Species Act in the four southeastern U.S. states where it lives. Rarity of this species is thought to be attributable in part to small population sizes and limited seed production. Protocols for in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed cryopreservation are presented that will assist in the safeguarding and augmentation of dwindling natural populations. Germination in vitro on growth regulator-free half-strength Murashige and Skoog (MS) medium after sterilization in H2O2 initiated the development of shoot cultures. Shoot multiplication and elongation occurred on half-strength MS salts containing 0.1 mg·L–l benzylaminopurine and 0.2 mg·L–l gibberellic acid, producing an average of 18 new shoots over a 6- to 8-week subculture cycle. Shoots rooted easily when planted into cutting mix after treatment with rooting powder containing indole-3-butyric acid (IBA) or in vitro rooting in medium with or without N-acetyl-L-aspartic acid (NAA). Plant survival after 1 month was 90% or higher for all treatments. Cryopreservation tests with seeds from three populations averaged 46.7% germination compared with control seed (no cryostorage) germination of 43%; differences were not statistically significant. Fresh seeds and seeds equilibrated for 1 to 4 weeks at room temperature and 12% relative humidity did not differ significantly in germination post-cryopreservation. Initial observations suggest that Georgia aster rapidly loses seed viability over 1 to 2 years when stored at room temperature. The ability to increase seed longevity through cryopreservation storage may be a critical step in the conservation of this species.

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Neil O. Anderson and Peter D. Ascher

Male and female fertility, seed germination, and progeny fertility were used to determine cultivar fertility in species of Lythrum. One short-, 11 mid-, and six long-styled cultivars were included in this study. Duplicates of several cultivars from different nurseries and three unknown cultivars from Minnesota gardens were also collected. Plants from 17 Minnesota and one Wisconsin population of L. salicaria served as fertile male and/or female testers. Pollen stainability (usually 100%) showed low levels of male gamete abortion. Pollen size within and among anther type varied widely; possible 2n gametes were present in primarily the short- and mid-anther morphs. Seed production per capsule from legitimate cross-pollinations, using cultivars as male parents with Minnesota or Wisconsin female testers, averaged 48 ± 36 across style morphs. Cultivars differed as males, as did anther morphs. With female fertility tests, seed set per capsule ranged from zero to 152 and averaged 54 ± 40 in legitimate pollinations (i.e., pollinations between stamen and styles of the same length). Seed set for other crosses showed similar trends. Only `Morden Gleam' produced no seed with all legitimate pollinations, although illegitimate selfs or interspecific crosses produced seed. Seed from legitimate crosses of L. salicaria × cultivars had 30% to 100% germination. Common male and female parents within each legitimate crossing group were not significantly different. This study showed that the cultivars are highly fertile when used as male or female parents with wild purple loosestrife, native species (L. alatum Pursh.), or other cultivars. Thus, cultivars grown in gardens could serve as pollen or seed sources for the continued spread of purple loosestrife. The implications of cultivar fertility, especially interspecific F1 hybrids, is discussed in relation to the spread of noxious weeds in wetlands.

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Mark W. Farnham

Using anther culture to generate doubled-haploid (DH) homozygous lines for use as parents in F1 hybrid crosses has become a common practice in breeding broccoli (Brassica oleracea L. Italica Group). During anther culture and subsequent embryogenesis and plant regeneration, polyploidization of microspore-derived embryos may not occur or it may occur accompanied by a doubling, tripling, quadrupling, octupling, or irregular polyploidization of the genome. Thus regenerants from the process can be haploids, diploids, triploids, tetraploids, octaploids, or aneuploids. The objectives of this research were to 1) conduct repeat cycles of broccoli anther culture using a group of F1 hybrids as anther donors and develop populations of regenerants; 2) analyze resulting populations using DNA flow cytometry and determine the influence of F1 source on frequency of different ploidy levels among regenerants; and 3) compare seed set in broccoli inbreds developed in a traditional selfing program compared to seed set in DH broccoli derived from anther culture. In two cycles (1994 and 1995) of anther culture, anther-derived populations of regenerants were developed using the F1 hybrids `Marathon', `Everest', `High Sierra', and `Futura' as sources of anthers. In 1994, `Everest', `High Sierra', and `Futura' yielded populations that included 2% to 7% haploids, 53% to 56% diploids, 32% to 38% tetraploids, and 5% to 6% other types. `Marathon'-derived regenerants were 5% haploid, 78% diploid, 15% tetraploid, and 2% other, showing significantly more diploids. In 1995, `Marathon' regenerants again included significantly more diploids and fewer tetraploids than those derived from other F1 sources, confirming that the genotype of the anther source affects the frequency of a particular ploidy level among regenerants derived from culture. In manual self-pollinations of 1994 regenerants, only diploids and rare tetraploids set seed. When plants that set no seed were discounted, seed production following manual self pollinations of 1995 regenerants was not significantly different from that of traditional inbreds derived from the same F1 sources.

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M.J. Havey

The primary source (S cytoplasm) of cytoplasmic-genic male sterility (CMS) used to produce hybrid-onion (Allium cepa L.) seed traces back to a single plant identified in 1925 in Davis, California. Many open-pollinated populations also possess this cytoplasm, creating an undesirable state of cytoplasmic uniformity. Transfer of cytoplasms from related species into cultivated populations may produce new sources of CMS. In an attempt to diversify the cytoplasms conditioning male sterility, the cytoplasm of Allium galanthum Kar. et Kir. was backcrossed for seven generations to bulb-onion populations. The flowers of galanthum-cytoplasmic populations possess upwardly curved perianth and filaments with no anthers, making identification of male-sterile plants easier than for either S- or T-cytoplasmic male-sterile onion plants. Mean seed yield per bulb of the galanthum-cytoplasmic populations was measured in cages using blue-bottle flies (Calliphora erythrocephala Meig.) as pollinators and was not significantly different from one of two S-cytoplasmic male-sterile F1 lines, a T-cytoplasmic male-sterile inbred line, or N-cytoplasmic male-fertile lines. Male-sterile lines possessing either the S or galanthum cytoplasm were each crossed with populations known to be homozygous dominant and recessive at the nuclear locus conditioning male-fertility restoration of S cytoplasm and progenies were scored for male-fertility restoration. Nuclear restorers of male fertility for S cytoplasm did not condition male fertility for the galanthum-cytoplasmic populations. It is intended that these galanthum-cytoplasmic onion populations be used as an alternative male-sterile cytoplasm for the diversification of hybrid onion seed production.