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  • Author or Editor: Pablo Jourdan x
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Budgetary, logistical and time constraints frequently limit the extent of hands-on activities included in introductory courses in horticulture. Model organisms can facilitate the demonstration of broad-based principles and enhance learning. The Wisconsin Fast Plants®, rapid cycling forms of Brassica rapa, represent such a model organism with diverse and useful applications in horticultural instruction. These organisms offer not only a practical advantage for teaching, but also facilitate learning at higher levels of thinking because they permit integration of concepts at the molecular, organismal, and population levels. A large collection of educational activities is currently available with these plants that can be used to enhance understanding of basic botanical principles; many aspects of plant physiology; broad principles of crop production; genetics and plant breeding; and molecular manipulations. The educational strategy afforded by these plants will be described.

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The regal pelargonium (P. x domesticum) is generally characterized by low fertility and poor seed set. In studys designed to assess factors that contribute to low fecundity in this crop we have examined genotype interactions among various cultivars and have identified lines that differ in degree of male and female fertility.

The objective of this study was to examine genotypic variation, other than self-incompatibility, of P. x domesticum pistils in supporting the development of the male gametophyte. Variation in pollen germination and growth was assessed after crossing either a male of high fertility or a mate of poor fertility to nine different selections of varying female fertility. Styles were harvested 2 hours after pollination and examined using fluorescence microscopy to determine the number of germinated pollen grains on the stigma and the number of pollen tubes growing down the style.

Female selections displayed large differences in their ability to support pollen tubes. Styles from different females pollinated with the same male varied in average number of pollen tubes from 30 to 2.

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Phlox is an important genus of herbaceous ornamental plants previously targeted for germplasm development, characterization, and enhancement by the U.S. Department of Agriculture, National Plant Germplasm System. Among Phlox in cultivation, Phlox paniculata is the most widely grown and intensively bred species, but little is known about variation in genome size and ploidy of this species or of related taxa that may be used for germplasm enhancement. The objective of this study was to assess cytotype variation in a diverse collection of cultivars and wild germplasm of P. paniculata (subsection Paniculatae) and of related taxa in subsections Paniculatae and Phlox. The collection included 138 accessions from seven species and two interspecific hybrids. Flow cytometry was used to estimate holoploid (2C) genome sizes and to infer ploidy levels. Chromosome counts were made to calibrate ploidy with genome size for a subset of taxa. Most cultivars were diploid (2n = 2x = 14) and had mean genome sizes that did not vary between subsections Paniculatae (14.33 pg) and Phlox (14.23 pg) although size variation was greater among cultivars within subsection Phlox. Triploid cultivars of P. paniculata, with a mean genome size of 21.36 pg and mitotic chromosome counts of 2n = 3x = 21, were identified. Such triploids suggests previous interploid hybridization within this taxon. Five tetraploid (2n = 4x = 28) cultivars were found in subsection Phlox; all were selections of P. glaberrima ssp. triflora, and had a mean genome size of 25.44 pg; chromosome counts in one of these confirmed they were tetraploid. The putative hybrid Phlox Suffruticosa Group ‘Miss Lingard’ showed an intermediate genome size of 21.21 pg supporting a triploid, hybrid origin of this taxon. Mean 2C genome sizes among wild-collected accessions were similar to values reported for cultivars (Paniculatae = 14.59 pg, Phlox = 14.23 pg), but taxa in subsection Phlox exhibited greater variation that included two tetraploids identified among wild-collected accessions; one, of P. pulchra, had a mean genome size of 26.17 pg, representing the first report of polyploidy in the taxon. This is the first report on genome size for the majority of species in the study. Although genome size could not be used to differentiate taxa in subsections Paniculatae and Phlox, the data provide further insights into cytotype variation of Phlox germplasm useful for plant breeders and systematists.

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Brassica napus (genome aacc), a natural allotetraploid derived from hybridization between B. oleracea L. (genome cc) and B. rapa L. (genome aa), was synthesized by sexual and somatic interspecific hybridizations from the same parent plants to compare the two methods of combining genomes and assess the genetic consequences of bypassing the gametophytic phase before hybrid formation. Highly heterozygous species parents were first produced by intraspecific hybridization between two subspecies each of B. oleracea and B. rapa. Leaf tissue from young plants of both parental species served as a source of protoplasts for fusion; the same plants were later used for crosses. Seventy-two somatic hybrids were produced using a polyethylene glycol-mediated fusion protocol and 27 sexual hybrids were obtained by embryo rescue. Somatic hybrids were produced between one B. oleracea and two sibling B. rapa plants. Sexual hybrids were successfully produced with only one of the two B. rapa siblings. Hybrids were identified by morphology, isozyme patterns, and total DNA content. Although fertile allotetraploid somatic hybrids were obtained within 7 months after seeding parent lines, >1 year was required to produce fertile sexual hybrids.

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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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Before attempting transformation of Pelargonium spp., it is necessary to develop procedures for shoot regeneration under selective conditions (kanamycin resistance). Leaf disks of various cultivars of regal, zonal, ivy-leaved, and scented pelargoniums were compared for shoot regenerability and phenolic production. Disks were cultured on MS medium supplemented with 10.7 μM NAA and 8.9 μM BA. After three weeks in darkness, each disk was transferred to MS with 0.89 μM BA and evaluated for proportion of disks with callus, extent of disk development, and the rate and intensity of phenolic halo production. Phenolic production was not correlated with shoot regeneration ability. Regal pelargoniums had low phenolic production, while ivy-leaved pelargoniums had both the highest phenolic production and callus formation. Shoot regeneration from leaf disks was inhibited on media containing 100 μg/ml kanamycin.

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The Wisconsin Fast Plants® (rapid cycling forms of Brassica rapa) have become a major educational tool in basic botany, plant physiology and plant genetics courses because of their versatility, ease of manipulation, and short life cycle (40-day generation). Various educational activities already developed by the Fast Plants Network can be incorporated effectively into established horticulture courses. We have been utilizing these plants in some of our introductory courses and have developed additional protocols and materials. Some of the new educational activities utilize the rapid cycling form of B. oleracea, Examples of these materials will be presented, including: new genetic stocks and use of isoenzyme markers; use of growth regulators (e.g., uniconazole) to modify plant growth and development; post-harvest physiology (e.g., growth, chlorophyll and starch content); hydroponics and plant nutrition; cell and tissue culture (micropropagation, anther culture, protoplast culture, transformation); and biochemical studies (e.g., enzyme isolation and characterization, extraction and characterization of DNA, etc).

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Poor seed set in P. xdomesticum hinders development of new and improved cultivars. Crossing data has indicated differences among selections in male fertility. A study was undertaken to examine the amount of male fertility in the species as evidenced by pollen staining and pollen germination and development. Eight P. xdomesticum selections were crossed as males onto two selections used as females. Styles were harvested 2 hours after pollination and examined using fluorescence microscopy to determine the number of germinated pollen grains on the stigma and the number of pollen tubes produced in the style. Pollen from the 8 selections was tested for viability by staining with fluorescein diacetate (FDA) and examined using fluorescence microscopy. The males varied in pollen viability from 69% pollen staining producing 16 pollen tubes to 18% saining producing 3 tubes. The most fertile males produce enough pollen tubes to effect fertilization of all of the ovules in a P. xdomesticum ovary.

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Abstract

More than 65 different genotypes, including cultivars and inbred lines, from five cruciferous species (Brassica oleracea L., B. campestris L., B. napus L., B. juncea L., and Raphanus sativus L.) were tested for their in vitro response of leaf protoplasts. Protoplasts were cultured in three liquid media and the resulting colonies were placed on seven test regeneration media. Significant differences among the species were found in plating efficiency in the frequency of shoot regeneration. Two broad response groups were identified: 1) Cultivars from B. oleracea and B. napus—these generally yielded protoplasts that were able to divide, form colonies at high frequencies, and regenerate shoots at variable frequencies; and 2) cultivars of the other species evaluated, which typically exhibited low plating efficiencies and little, if any, shoot regeneration. Evaluation for the effect of the cytoplasmic constitution of a few B. oleracea breeding lines on in vitro performance indicated that protoplasts carrying the Ogura (R1) male-sterile cytoplasm regenerated shoots at slightly lower frequencies than the corresponding alloplasmic-fertile lines. Genotypes exhibiting high frequency of shoot formation in one medium also had efficient shoot regeneration in other media as well, while genotypes with low shoot regeneration responded consistently in the different media used. This consistency in response indicates that genotype plays a critical role in determining the success of leaf protoplast culture in the crucifers.

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As a prelude to interspecific hybridization, we compared the floral biology of bottlebrush buckeye (Aesculus parviflora) and red buckeye (A. pavia) by examining inflorescence morphology, pattern of floral anthesis, sex expression, and the effects of panicle decapitation on complete flower development. Inflorescences of both species (n = 1606) were randomly selected and analyzed for length, total number of flowers and complete flower number and location. The pattern of anthesis was observed in four genotypes using 10–30 inflorescences per plant. For each flower, its date of anthesis, position on both the rachis and cincinnus, and sex were recorded. For studies of panicle decapitation, sets of panicles were selected and one member was severed in half early in development in an attempt to increase the number of complete flowers. More than one-fourth of all panicles observed were completely staminate. For both species, the ratio of complete flowers to male flowers (C:M) within mixed panicles was about 5%. Complete flowers were observed in the basal portion of A. pavia inflorescences and in the apical portion of A. parviflora inflorescences. Anthesis progressed from base to tip over a period of 6–11 days. Complete flowers are present in A. pavia from the beginning of anthesis but do not appear in A. parviflora until the fifth day of anthesis. Staminate flowers are present throughout anthesis in both species. Severing panicles in half increased the potential for differentiating complete flowers. In conclusion, the frequency of complete flowers in both species was quite low, but could be increased by panicle decapitation to increase opportunities for controlled hybridization.

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