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A method was devised for infecting Anthurium andraeanum Linden ex André, an economically important ornamental monocot, with Agrobacterium tumefaciens. Tumors were obtained on plant stems 7 to 10 weeks after inoculation with oncogenic A. tumefaciens strains C58 and A281 cultured previously in an induction medium containing 200 μm acetosyringone at pH 5.5. A higher percentage of tumors were formed in vitro on etiolated internodes (32%) than on green leaf (2%) or petiole explants (3%) 4 weeks after inoculation with induced C58. All explants treated with nontumorigenic A. radiobacter or with induction medium alone failed to produce tumors. Chromatograms showed an accumulation of nopaline in internode explant tumors induced with C58. DNA amplification and hybridization studies showed that the DNA from these tumors, but not from noninoculated anthurium tissue, contained sequences homologous to the nopaline synthase gene of A. tumefaciens T-DNA. Chemical names used 3,5-dimethoxy 4-hydroxyacetophenone (acetosyringone).
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.
Dendrobium `Uniwai Mist' (UH800) protocorm-like bodies (plbs) infected by Cymbidium mosaic virus (CyMV) were grown in Vacin and Went liquid medium supplemented by 15% coconut water with one of four antiviral compounds: ribavirin (virazole), cycloleucine, 3-deazauridine, and dithiouracil. After 5 weeks of treatment, 0.2 mM dithiouracil reduced CyMV to non-detectable levels and 0.05 mM 3-deazauridine lowered CyMV concentration by 66%. Both 0.2 mM dithiouracil and 0.05 mM 3-deazauridine resulted in smaller, darker green plbs and ∼10% dead tissue as compared to the control. Virus concentration resumed to previous levels by 5 weeks after treatment with dithiouracil was discontinued. Ribavirin at 0.1 mM lowered virus concentration by 50% five weeks after treatment was stopped. 0.05 mM and 0.15 mM dithiouracil and 0.01 mM, 0.05 mM, 0.08 mM, 0.5 mM, and 0.75 mM ribavirin had no effect on virus concentration. 0.5 mM and 0.75 mM ribavirin resulted tissue death.
Leaf explants of seven cultivars of Hawaiian anthuriums (Anthurium andraeanum Linden ex André cv. Kaumana, Kozohara, Marian Seefurth, Mauna Kea, Nitta, Ozaki, and Paradise Pink) produced callus most successfully after 2 to 3 months on a modified Pierik medium containing 0.36 μm 2,4-D and 4.4 μm BA. Petiole explants callused best on Pierik modified Pierik, and Finnie and van Staden media. Long-term cultures of callus from Univ. of Hawaii anthurium selections UH965, UH1060, and UH1003 were maintained for 12 to 13 months and were still capable of plantlet regeneration. Adventitious plantlets were recovered from callus plated on a Kunisaki medium containing 2.2 or 22 μm BA. Regeneration appeared to be organogenic rather than embryogenic and varied among the genotypes tested. Chemical names used: N- (phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D).
Flow cytometry (FC) has proven to be an efficient and reliable method to estimate nuclear DNA content (genome size) in quantifiable units useful for genetic and molecular biology studies. This method also makes possible determination of the variation in nuclear DNA content between related taxa, which gives insights into the process of speciation. In this study, DNA content was determined in nuclei isolated from leaves of 21 Dendrobium species representing each of the major taxonomic groups used in the Univ. of Hawaii breeding program. Nuclei were mechanically isolated, stained with the nucleic acid-specific fluorochrom propidium iodide, and DNA content determined using a Coulter Epics 753 laser flow cytometer. Chicken erythrocyte nuclei (2C = 2.33 pg DNA) were used as an internal standard for direct comparative measurement. The mean diploid genome (2C) values for Dendrobium species ranged from 3.36 to 5.06 pg. Genome sizes were evaluated for possible use as discrete characters for taxonomic group assignment and compared to previous data on breeding compatibility and evolutionary relationship between species.
Burrowing nematode, Radopholus similis, reduces flower-yield-infected anthurium fields. Genetic resistance is one alternative to chemical control of the disease in anthurium. Seventeen commercial anthurium varieties, established in vitro on anthurium nutrient medium, were inoculated with burrowing nematodes to screen for tolerance. Three months after inoculation, plant responses were compared by number of nematodes recovered and by symptom index and plant weight loss with respect to non-inoculated plants. Results show that `Mauna Kea' and `Flamingo' anthuriums are among the most tolerant, while `Ozaki' is one of the most susceptible. These results are consistent with grower field evaluation. Nematode count is positively correlated with symptom index and weight loss. The mechanism of tolerance or resistance of anthurium toward burrowing nematode is unclear. However, due to the fact that burrowing nematode is a migratory endoparasite, a preinfectional resistance or tolerance mechanism is more likely to take place.
Flowers emit volatile compounds that attract pollinators. In ornamental plant breeding programs, fragrance is a significant character that adds value to flowers for its consumer appeal. In Hawaii, anthurium (Araceae) is an important crop used for cut flowers and flowering potted plants. Unlike other ornamentals, fragrance is not presently associated with commercial anthuriums. However, several anthurium species are known to have distinctive scents. To obtain the novelty trait of fragrance in anthurium, an understanding of anthurium scent genetics, physiology, and chemistry is required. Scented anthurium species and hybrids in the Univ. of Hawaii germplasm collection have been studied. Fragrance emission among species varies with time of day—some species being scented only in the morning, only at night, or all day long. Fragrance emission also varies with stage of spadix development, with some species having scent as pistillate and/or staminate flowers. The species sampled comprise five categories: A. amnicola, A. formosum, and A. lindenianum are minty; A. armeniense is sweet; A. gracile is floral; A. bicollectivum, A. cerrobaulense, A. folsomii, and A. harleyii are fruity; and A. supianum is fishy. Some of the chemical components are illustrated.
Two full-length cDNA clones, Den-CHS-4 and Den-DFR-1, encoding chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR) were obtained from flower bud RNA of a lavender cyanidin-accumulating Dendrobium Sw. hybrid using reverse transcription-polymerase chain reaction (RT-PCR). Northern analyses indicated that both genes are expressed in all developmental stages of buds, with highest expression in the medium-sized buds. RT-PCR analyses showed that DFR expression was confined to floral tissue while CHS was expressed in floral and vegetative tissues but not in pseudobulbs. The nucleotide sequence of a DFR clone isolated from a pale orange pelargonidin-accumulating Dendrobium hybrid was exactly the same as Den-DFR-1, ruling out the substrate specificity of DFR as a possible cause of the color difference.
Two cultivars of Anthurium andraeanum Hort. hybrids, `Paradise Pink' and `Tropic Flame', were transformed by Agrobacterium to contain gene sequences for Shiva-1, a cecropin-based lytic peptide. The antibacterial gene was driven by a 35-35S cauliflower mosaic viral (35-35S CaMV) promoter and the construct included the secretory signal sequence for pathogenesis-related protein 1b (PR1b). Blight tolerance of regenerated plants was tested by inoculation with a virulent strain of Xanthomonas axonopodis (formerly campestris) pv. dieffenbachiae (Xad) that is bioluminescent to allow detection of symptomless infections in Shiva-1 transformants. Primary regenerants for two Shiva-1 transgenic lines of `Paradise Pink' displayed significantly enhanced tolerance to bacterial blight over blight susceptible `Rudolph' and even the blight tolerant `Kalapana'. Two Shiva-1 transgenic lines of `Tropic Flame' showed no improved resistance when compared to the control at the mean percent leaf infection level. One Shiva-1 transgenic line of `Tropic Flame' was unexpectedly more susceptible to blight than the nontransgenic control. Low expression of Shiva-1 observed in this line is hypothesized to be the cause of its increased susceptibility to Xad.