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Joseph D. Postman, Kim E. Hummer, and Kirk W. Pomper

A pawpaw (Asimina triloba) regional variety trial (PRVT) was established at the U.S. Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository (NCGR), Corvallis, Ore., in Fall 1995. This orchard was a replicated planting of 28 commercially available varieties or advanced selections from the PawPaw Foundation (PPF; Frankfort, Ky.), with eight replicate trees of each selection grafted onto seedling rootstocks and planted in a randomized block design. Two years after planting, 32 trees had either failed to establish or had died after an initial healthy start. By July 1999, 25% of grafted trees had died due to a vascular wilt-like disease, and 2 years later mortality exceeded 50%. Grafted selections with the lowest symptom severity include 1-7-2, 2-54, 7-90, 8-58, 9-58, `Mitchell', `PA-Golden #1', `Taylor' and `Wilson'. Seedling guard trees were unaffected until July 2000, when six guard trees of 76 died and 10 more were declining. By July 2001, 14 guard trees were dead. No fungi were consistently isolated from declining trees. A number of bacteria were isolated from infected trees, but no specific pathogen has been confirmed as the causal agent. Polymerase chain reaction (PCR) tests for phytoplasmas and for the bacterium Xylella fastidiosa were also negative. Research is ongoing to determine if a bacterial pathogen was the cause of the pawpaw decline in the Oregon PRVT.

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S. Brooks Parrish, Renjuan Qian, Sandra B. Wilson, Gary W. Knox, and Zhanao Deng

and analysis. Just before anthesis, flowers were opened and 12 anthers from three inflorescences were collected in microcentrifuge tubes. One hundred microliters of cotton-blue stain was added to the anthers and they were placed in a 65 °C water bath

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Arthur Villordon, Christopher Clark, Don LaBonte, and Nurit Firon

measured using ImageTool (Univ. of Texas Health Science Center at San Antonio, available from < >). Entire node sections with non-emerged roots were excised and subjected to trypan blue staining ( Desmond et al., 2008 ) to

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Todd A. Russell, Joseph B. Morton, and Bradford Bearce

Nursery liners of Viburnum carlesii, Hemsl; Acer palmatum, Thunb., cv. 'Bloodgood'; and Buxus sempervirens L., cv. 'Vardar Valley' were planted in several root media at three P levels which had been inoculated or not inoculated with species of endomycorrhizal fungi. Analysis of extent of root infection (Trypan Blue staining procedure) revealed no root infection or colonization of any of the three species in any root medium. However, maples grown in an inoculated peat:sand (1:1, v/v) medium at .024 g P/liter grew taller than maples in the same non-inoculated medium at .24, .024, or .0024 g P/liter of medium. Mean height of maples in inoculated media were greater than heights of maples in non-inoculated media when height data from all three P levels in each medium were combined. Infection of Sorghum Sudanese, (Piper) Stapf, root: was inhibited when media was amended with composted or noncomposted pine bark but was not inhibited by pine bark leachates.

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Wenhao Dai, Zong-Ming Cheng, and Wayne Sargent

Transgenic hybrid aspens (Populus tremuloides × P. tremula) were produced by Agrobacterium-mediated transformation and confirmed by polymerase chain reaction. Three promoters (CaMV 35S, Heat shock, and Rol C) were used to drive transcription of chimeric genes -glucuronidase (GUS), npt-II, and rol B. Stem sections in ≈100 mm thick, leaf blades, and root tips of transgenic aspen were treated with X-Gluc solution for 2 to 12 h in a 37 °C incubator and fixed in a solution containing 5% formaldehyde, 5% acetic acid, and 20% ethanol (FAA) for 10 min. After washing with 50% ethanol twice and clearing with absolute ethanol until free of chlorophyll, the GUS expression (localization and intensity of blue staining) in leaf, stem, and root at different growth stages were evaluated and photographed under the light microscope. When CaMV35S and rol C were used as promoters, the GUS gene was expressed in all parts of mature stem except pith, with the strongest activity in phloem. The heat shock promoter gave rise to very strong expression only in epidermis and phloem. In the young stem, GUS activity was detected in epidermis, parenchyma, vascular cambium, and primary xylem in CaMV35S-GUS transformed aspen shoots. The rol C promoter produced GUS gene expression in all stem tissues. When the heat shock promoter was used, the GUS gene expressed in a more tissue-specific manner, especially in mature stems, with activity mainly in parenchyma. In young leaf tissues, the GUS activity was primarily located in veins and mesophyll. In the mature leaves, no blue staining was found in the main vein. In root tip, the GUS gene driven by CaMV35S and heat shock promoters were expressed in the columella, vascular, and root apical meristem with very strong expression in the root apical meristem. Aspen plants transformed by rol C-Gus construct showed less or no expression in the columella.

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Desmond G. Mortley

Greenhouse studies were conducted to evaluate 5 levels of Mn (0.00025 to 0.1 g.L-1) on Mn toxicity or tolerance of sweetpotato [Ipomoea batatas (L.) Lam] grown in a modified half Hoagland's solution. The presence of oxidized Mn on the roots and leaves was demonstrated by the blue staining test with benzidene and the solubility and bleaching of oxidized Mn in the oxalic-sulfuric acid solution. Both storage root and foliage fresh and dry weights were highest at Mn concn of 0.00025 g.L-1 in the nutrient solution, while fibrous root dry weight was highest with 0.01 g.L-1 Mn in the solution. More Mn accumulated in foliage than in fibrous roots for all levels of Mn evaluated. N, P, and K concn in foliage was highest at a Mn concn of 0.1 g.L-1 Mn in the solution. Foliage dry weight was preserved up to a high Mn level of about 2700 ug. g-1 Mn in tissues, while taht for storage roots was preserved up to a high Mn level of about 1000 ug. g-1 in the tissues. Deposition of oxidized Mn was observed on fibrous roots particularly at the highest Mn levels in the nutrient solution.

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Haiyan Xu, Folian Li, Yuezhi Pan, and Xun Gong

The investigation of hybridization processes and embryogenesis of heterozygote is an effective approach for early hybrids’ identification, which could provide reliable information for successful crossbreeding. In this study, we reported the whole hybridization processes of the direct cross and reciprocal cross between Michelia yunnanensis Franch. ex Finet et Gagnep. and Michelia crassipes Law using fluorescence microscopy after aniline blue staining, with the pollen germination on stigmas, pollen tube growth in styles, and subsequent extension into the embryo sac as well as the double fertilization processes are documented in detail. The M. yunnanensis × M. crassipes combination displayed considerable cross-compatibility, and the heterozygote embryogenesis was further observed with an approach of modified cryosectioning technique. Besides, the whole formation processes of hybrid seeds from artificial pollination to maturation were successfully observed. However, in the reciprocal cross, we found incompatibility between pollen grains of M. yunnanensis and stigmas of M. crassipes for the reason of hysteretic identification, as well as the abnormal callose deposition which belongs to the prefertilization barriers. This is the first study in which the complete and clear hybridization processes in Michelia were reported. We inferred that unilateral incompatibility of M. crassipes detected in this study may also exist in some other Michelia species. In artificial hybridization practices, we suggest some special treatments for overcoming prefertilization barrier should be taken when treating M. crassipes as the maternal parent.

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Zhanyuan Zhang, Dermot P. Coyne, and Amitava Mitra

Factors influencing Agrobacterium tumefaciens-mediated transformation of common beans (Phaseolus vulgaris L.) were examined using an intron-containing β-glucuronidase (GUS) gene as a reporter system to develop a repeatable transformation protocol. Tissue culture procedures used were based on direct shoot organogenesis. Two A. tumefaciens strains—A2760 and EHA105—were used, with emphasis on the former due to its overall higher infection rate. Eleven common-bean genotypes were compared for susceptibility to strain A2760 or EHA105. The pinto bean `Othello' was used extensively in testing different transformation conditions. Factors significantly affecting transformation rate were Agrobacterium × host interactions, explant maturity, preculture and cocultivation conditions, and selection schemes, based on transient GUS gene expression. The best transformation conditions were the use of susceptible genotypes and explants derived from mature seeds, preconditioning of explants in a medium containing 20 μmol of benzyladenine (BA) in darkness or on a filter paper, dipping explants in high concentrations of Agrobacterium cell suspension (OD650 = 0.8-1.0) followed by a long-term (6-day) cocultivation period on a semisolid agar medium in the presence of cytokinin or 3-day cocultivation on a moistened filter paper, and the use of lethal levels of selective agents. About 4% of explants, or 14% of regenerated shoots or buds, were putatively transgenic, as indicated by GUS blue staining throughout the entire shoot or bud, after explants were transformed with Agrobacterium strain A2760 using an optimized protocol.

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Brad Geary, Jared Benson, Steven Wood, James Logan, Ben Brulotte, Alan Chambers, Jeff Maughan, and Mikel Stevens

Endophytic fungi that are classified into the genus Neotyphodium have developed into a very unique niche. Their specific host plants are the fescues and ryegrasses. Through fungal biosynthesis of secondary metabolites, the host plant receives several benefits. These benefits include resistance to insects such as aphids, chinch bug, and argentine stem weevil, increased drought tolerance, and increased competiveness. These secondary metabolites comprise four groups of alkaloids. The alkaloids are loline, peramine, Lolitrem B, and ergovaline. The quantitative alkaloid profile is unique for each isolate. The characterization of these endophytes is necessary for identification of specific isolates. We report the characterization of ten endophytic strains Lp1, Lp2, Lp3, Lm4, Lm5, Fp6, Fp7 Fp8, Fp9, Fp10. The characterization of each isolate includes: morphology, sporulation, growth rates, microsatellite fingerprint, and alkaloid profile. The isolated colonies bear resemblance to raised brain-like structures and are yellow to tan in color. Growth rates range between 0.1 and 0.25 mm/day. No colonies produced any form of sporulation. Fp6 was found to have the highest loline concentration of any isolate. AFLP analysis was performed on the isolates to test for relatedness. Distinct clades were formed and grouped by host. The main groups were those isolated from Lolium or Festuca varieties. Isolates Fp8 and Fp9 were most related to each other, and have also been found to be doubly infected. The double infection is described to be Phialophora-like, due to the presence of thin highly branched hyphae when observed under light microscopy with aniline blue staining.

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Guo-qing Song, Hideo Honda, and Ken-ichi Yamaguchi

-II promoter—β-glucuronidase expression. A typical pattern of CAB2 -p-GUS expression was present in all six transgenic lines. In 4-week-old plantlets grown in vitro under 16 h of 30 μmol·m −2 ·s −1 light, blue staining indicating that the CAB2 -p