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Jennifer A. Ehrenberger* and Adelheid R. Kuehnle

A hybridization strategy for certain coloration could be developed based on accurate histological information of parental material together with the knowledge of heritability of color and color intensity. A sample of 12 Anthurium species and hybrids were histologically examined for pigmentation in spathes using a new method employing vacuum infiltration of spathe tissue with polyethylene glycol (PEG) prior to cross-sectioning. PEG infiltration displaces intercellular air spaces between cells. This method greatly improved the clarity of the cross sections and consequently improved observations of spatial localization of anthocyanins and chloroplasts. This infiltration method accurately identified the spatial localization of pigments for future breeding reference, notably among Anthurium species.

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Katrina G. Porter and Adelheid R. Kuehnle

Our objective was to develop a virucide-based method to eliminate cymbidium mosaic virus from infected dendrobium protocorm-like bodies during micropropagation. We tested two virucides—dithiouracil and ribavirin. The best treatments for recovery of virus-negative tissue and subsequent plant development were 0.1 mm ribavirin or 0.2 mm dithiouracil in liquid medium for 5 or 6 weeks followed by culture on solid medium with 0.1 mm ribavirin or 0.1 mm dithiouracil, respectively. Most propagules transferred to solid medium were virus-free at 17 weeks on ribavirin, with up to 100% free of virus after 32 to 38 weeks on ribavirin or dithiouracil. Virus loss among protocorm-like bodies cultured without virucides ranged from 0% to 37%. Most plants remained free of virus 2.5 years after chemotherapy and flowered normally.

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Fure-Chyi Chen and Adelheid R. Kuehnle

Several procedures were tested in development of a gene transfer protocol for anthurium. Etiolated internode segments of anthurium cultivars `Rudolph' and `UH1060' were co-cultured with Agrobacterium tumefaciens LBA4404 carrying the chimeric genes neo, for antibiotic resistance, and att encoding antibacterial attacin. Assays of genomic DNA and RNA from kanamycin-resistant `Rudolph' and DNA from `UH1060' plantlets, recovered as soon as 1 year after culture on selection media, indicated the presence of introduced genes, including neo and att, and transcription of att. Western analysis confirmed the expression of attacin protein in calli induced from laminae of regenerated kanamycinresistant `Rudolph' plantlets. Use of tobacco nurse cells during co-cultivation of internodes with Agrobacterium did not increase recovery of shoots under the regeneration conditions used. Improvements in culture and antibiotic selection conditions during plant development are suggested.

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Rasika G. Mudalige and Adelheid R. Kuehnle

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Tridate Khaithong, Brent S. Sipes, and Adelheid R. Kuehnle

Lack of a conclusive evidence of ingestion of plastid components by plant-parasitic nematodes cautions the use of plastid transformation technology for nematode resistance. Nematode-resistant effector proteins generally require ingestion by the nematode to be effective. Transgene-encoded proteins produced in plastids are not known to be exported into the cytoplasm. Disintegration of plant cell organelles after nematode feeding suggests that nematodes possibly ingest plastid components. Proof of ingestion will validate the development of plastid transformation for nematode resistance. Small subunit ribulose-1,5-bisphosphate carboxylase/oxygenase (SSRubisco) protein is prevalent in chloroplasts and thus chosen as a study molecule. The migratory endoparasitic nematodes Pratylenchus penetrans and Radopholus similis were cultured on green carrot callus containing chloroplasts. Total nematode proteins were extracted and subjected to western blot analysis using cross-reactive polyclonal antibody raised against spinach SSRubisco. Positive detection of SSRubisco occurred in protein extracts from nematodes fed on green carrot callus, but not in extracts from nematodes cultured on non-green alfalfa root callus as negative control. These results confirm the ingestion of plastid components of MW 14 kDA by migratory endoparasitic nematodes.

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Wang Koon-Hui, Brent S. Sipes, and Adelheid R. Kuehnle

Radopholus similis distribution in Anthurium plant tissue was determined in a greenhouse experiment. Two thousand mixed life stages of R. similis per plant were inoculated onto Anthurium cultivars `Alii' and `Midori'. Nine months later, nematodes per gram of tissue were determined from stem sections (0-3, 3-6, and above 6 cm from the base), the lowest leaf petiole, and root tissue. R. similis occurred in all stem sections, leaf petioles, and roots in both `Alii' and `Midori'. Nematode distribution differed between the two cultivars. `Midori' had higher numbers of nematode in the roots whereas `Alii' had higher numbers of nematode in the stem sections and first leaf petiole. Anthurium apical stem cuttings could be contaminated with R. similis and may not be a nematode-free propagation material.

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Kullanart Obsuwan, Wayne B. Borth, John Hu, and Adelheid R. Kuehnle

A Cymbidium mosaic virus movement protein gene with a site-specific mutation (mut11) under control of a ubiquitin promoter was inserted using biolistics into two Dendrobium varieties with the intention of creating CymMV-resistant orchids. Presence of the transgene in regenerated plants of D. × Jaquelyn Thomas `Uniwai Mist' and D. x Jaq–Hawaii `Uniwai Pearl' was confirmed by PCR using genomic DNA, and mut11-positive plants were potted ex vitro. Forty-two transgenic plants and four non-transgenic control plants at the 4- to 6-leaf stage were inoculated with a 1:1000 dilution of CymMV obtained from infected orchids. Plants were analyzed for systemic infection using tissue blot immunoassay (TBIA). Seventeen plants from at least six independent transformations remained virus-free, whereas all control plants were infected with CymMV within 1 month. Further analysis by RT-PCR showed that the mut-11 mRNA was detectable in only two of these 17 plants. All plants were challenged again with a second CymMV inoculation as above, followed by TBIA analysis after 1 month. Thirteen of 17 plants remained free from virus. A third challenge of these plants with CymMV as above was followed by TBIA analysis at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 12 months after challenge. Results at 2 weeks post-inoculation showed that all six controls and four individual transgenic plants, including the RT-PCR-positive plants, became systemically infected. Nine transgenic plants from both varieties remained free from CymMV 12 months after the third challenge. Lack of detectable mut11 mRNA in these resistant lines suggests that a post-transcriptional gene silencing (PTGS) mechanism may be conferring resistance to CymMV.

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Stacie L. Aragon, Keng-Chang Chuang, and Adelheid R. Kuehnle

Isolation of high quality nucleic acids from aroids can be difficult due to the presence of carbohydrates, phenolics, and other compounds that bind to and/or co-precipitate with the DNA or RNA. Methods previously used for marine algae, mango, and papaya were modified and successfully used for the simultaneous isolation of high quality genomic DNA and RNA from Anthurium, Colocasia, and Spathiphyllum leaves. Genomic DNA yields averaged 477 μg·g-1 fresh weight for Anthurium and 322 and 177 μg·g-1 fresh weight, respectively, for Colocasia and Spathiphyllum. Total RNA yields averaged 129 μg·g-1 fresh weight for Anthurium and 61 and 50 μg·g-1 fresh weight, respectively, for Colocasia Spathiphyllum. This method may be useful in co-isolating high quality nucleic acids from additional aroids and other plants.

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Tracie K. Matsumoto, David T. Webb, and Adelheid R. Kuehnle

Histological analysis of somatic embryos derived from in vitro-grown lamina of Anthurium andraeanumshowed bipolarity with the presence of shoot and root poles connected by procambium. Vascular connections between the explant and somatic embryos were not observed. Storage of proteins, starch and raphides as well as a suspensor-like structure and an epidermis were observed in the somatic embryos. Origin of the somatic embryos was from a proembryonic cell complex or possibly from a single cell by direct embryogenesis. Both modes of somatic embryogenesis arose from the mesophyll.

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Rasika G. Mudalige, Adelheid R. Kuehnle, and Teresita D. Amore

Perianths of 34 Dendrobium Sw. species and hybrids were examined to elucidate the roles of pigment distribution and shape of upper epidermal cells in determining color intensity, perception, and visual texture. Color intensity was determined by the spatial localization of anthocyanin in tissue layers, i.e., in the epidermal, subepidermal, and mesophyll layers, as well as by distribution of pigmented cells within the tissue layer. Anthocyanins were confined to the epidermal layer or subepidermal layer in flowers with low color intensity, whereas they were also in several layers of mesophyll in more intensely colored flowers. Striped patterns on the perianth were due to the restriction of pigment to cells surrounding the vascular bundles. Color perception is influenced by the presence or absence of carotenoids, which when present, were distributed in all cell layers. Anthocyanins in combination with carotenoids resulted in a variety of flower colors ranging from red, maroon, bronze to brown, depending on the relative location of the two pigments. Four types of epidermal cell shapes were identified in Dendrobium flowers: flat, dome, elongated dome, and papillate. Epidermal cell shape and cell packing in the mesophyll affected the visual texture. Petals and sepals with flat cells and a tightly packed mesophyll had a glossy texture, whereas dome cells and loosely packed mesophyll contributed a velvety texture. The labella in the majority of flowers examined had a complex epidermis with more than one epidermal cell shape, predominantly papillate epidermal cells.