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Ruth C. Martin, Machteld C. Mok, and David W.S. Mok

Cytokinins are widely used in tissue culture and transformation systems; however, little is known of their mode of action or the mechanisms regulating their levels in plant tissues. We are studying enzymes responsible for the metabolism of zeatin in immature seeds of Phaseolus. Selective expression of genes encoding such enzymes may regulate the level of active cytokinins during seed development as well as in in vitro systems. A zeatin O-xylosyltransferase, which mediates the formation of O-xylosylzeatin from trans-zeatin and UDP-xylose, has been isolated and monoclonal antibodies specific to the enzyme have been produced. Tissue print analyses demonstrated that the enzyme is primarily localized in the endosperm. ln situ localization and EM studies indicated that the enzyme is present in the cytoplasm and the nucleus. cDNA libraries were constructed from immature seed mRNA and immunopositive clones were selected. The products of these clones are being analyzed in E. coli and baculovirus expression systems.

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Shutian Tao, Danyang Wang, Cong Jin, Wei Sun, Xing Liu, Shaoling Zhang, Fuyong Gao, and Shahrokh Khanizadeh

-time polymerase chain reaction (qRT-PCR). Gene cloning and sequencing of PCR products. The corresponding nucleotide sequences of C4H in the National Center for Biotechnology Information (NCBI) database from plants in the Rosaceae family were obtained. For the

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Jiahua Xie, Todd C. Wehner, and Mark A. Conkling

Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes.

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Cheryl R. Hampson and Anita N. Azarenko

Self-incompatibility, a genetic mechanism enforcing out crossing, is most commonly controlled by a single, multi-allelic gene, designated the S-gene. Sporophytic self-incompatibility (SSI), a form of incompatibility determined by the parent plant rather than the gametes, is present in the Brassicaceae, Compositae and other families, and also in hazelnut (Corylus avellana L.). Little is known about the molecular basis of SSI in plants other than crucifers. An S-gene cloned from Brassica oleracea (donated by Dr. June Nasrallah, Cornell University) was used to probe genomic DNA obtained from seven hazelnut genotypes. DNA hybridization was observed in cultivars `Hall's Giant' and `Willamette'. Gene similarity was estimated to be 70-80%.

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Xian Shen*, Ling Guo, and Zhenlin Wei

Malus hupenensis var. `Pingyitiancha' is an important apple stock with many good characteristics, including waterloggig resistance, cold resistance, salt resistance and so on. The three group gene-HVA1 come from barley was transformed into `Pingyitiancha' mediated by Agrobacterium tumefaciens and transformed regeneration plants were obtained in this research. The HAV1 gene cloned from plasmid containing it (offered by Dr. Guo Weidong) by PCR with high fidelity pfu Taq DNA polymerase. It was ligated between BamH 1 and Sac 1 site in PUC118 vector, and identified by electrophoresis after digested with BamH 1 and Sac 1. Through nuclear sequence detecting, it is confirmed that the HAV1 gene cloned in this research is 703bp.This fragment was ligated with 11kb fragment from pB121 plasmid and constructed pBHA vetor. The pBHA vector was introduced in A.tum LBA4404 by triparental mating and the binary vector was obtained. It is cinfirmed that HVA1 gene had been insert in T-DNA by in situ hybirdization. Using `Pingyitiancha' shoot apex, mediated by A. tum. System, the HAV 1 gene was transformed into the plant. Kam resistance regeneration plants were obtained, 6 of them were confirmed as transformation plants by PCR and dot blot.

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Steven E. Lindow

Genes determining the ability of the bacterium Pseudomonas syringae to catalyze ice formation have been cloned and characterized. Ice nucleation active (Ice+) strains of this species are common on plants and the supercooling ability of frost sensitive plants is inversely proportional to the logarithm of the population size of Ice+ bacteria at temperatures above -5C. Recombinant Ice- derivatives off. syringae were produced by site-directed mutagenesis using deletion containing ice genes cloned form this species. The Ice- strains colonized potatoes well in field studies, reduced the population size of Ice+ bacterial strains by about 50-fold, and reduced the incidence of frost injury an average of 82% in several radiative frosts of temperatures in the range of -3 to -5 C. The ice gene has also been introduced into Solanum commersonii to determine its effect on increasing the tolerance of ice formation in this frost tolerant species. Transgenic plants exhibit a much higher threshold ice nucleation temperature than the parental plants.

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Daofeng Liu, Jing Ma, Jianfeng Yang, Tien V. Nguyen, Huamin Liu, Renwei Huang, Shunzhao Sui, and Mingyang Li

Wintersweet is a woody ornamental plant and has a long history of human cultivation. Few molecular markers have been characterized and remain scant in wintersweet. This study aimed to mine simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) from the transcriptomic database of wintersweet. A total of 3972 SSRs and 97,060 putative SNPs/indels (92,307 SNPs and 4753 indels) were identified in this data set. This study marks the highest number of SSR and SNP markers discovered to date from wintersweet by using transcriptome sequencing data. These identified markers will provide a useful source for molecular genetic studies such as genetic diversity and characterization, association mapping, and map-based gene cloning in wintersweet.

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Tong Geon Lee, Samuel F. Hutton, and Reza Shekasteband

Mechanization of farm work is increasingly demanded for the current system of fresh-market tomato (Solanum lycopersicum) production. One essential element for the adoption of mechanical harvest of fresh-market tomatoes is modification of plant architecture so that the crop can be grown without staking. To address this in the current production system, the stem length should be reduced. The tomato brachytic (br) locus has been shown to be a primary source of reducing stem length. To improve the effectiveness of marker-assisted selection (MAS) for the br-mediated trait and to provide resources for cloning this gene, we fine-mapped br to the tomato genome. Fine mapping of br to chromosome 1 was initiated by a survey of genome-wide single-nucleotide polymorphisms (SNPs) shown to be polymorphic between the br phenotype and normal using the tomato array, identifying the interval that harbors br. Genetic markers that flank the locus further permitted saturation of the interval. Twenty-six fixed homozygous recombinant lines were identified together in two different populations and tested with those markers. These efforts resulted in the first report that the br is fine-mapped to a 763-kb physical interval of tomato reference genome. The identified markers close to the br in the present study will be significant resources for MAS and gene cloning research.

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Bruce D. Whitaker and Gene E. Lester

Increases in phospholipase D [PLD (EC] and lipoxygenase [LOX (EC] activities are thought to play a critical role in senescence of mesocarp tissues in netted and nonnetted muskmelon (Cucumis melo L.) fruits. We have cloned and characterized two full-length cDNAs, CmPLDα1 and CmLOX1, encoding PLDα and LOX proteins in honeydew melon (C. melo Inodorus Group cv. Honey Brew). Relative levels of expression of the corresponding genes were determined by semi-quantitative RT-PCR in developing and mature fruit mesocarp tissues [20-60 d after pollination (DAP)], as well as in roots, leaves, and stems from 4-week-old and flowers from 6- to 7-week-old plants. The coding regions of CmPLDα1 and CmLOX1 cDNAs are, respectively, 2427 and 2634 nucleotides long, encoding proteins 808 and 877 amino acids in length. CmPLDα1 is very similar to PLDα genes from castor bean (Ricinis communis L.), cowpea (Vigna unguiculata L.), strawberry (Fragaria ×ananassa Duch.) and tomato (Lycopersicon esculentum Mill.) (77% nucleotide identity), and is the first PLD gene cloned from a cucurbit species. CmLOX1 has 94% nucleotide identity to a cucumber (Cucumis sativus L.) LOX gene expressed in roots and 80% identity to cucumber cotyledon lipid body LOX. In general, transcript of CmPLDα1 was much more abundant than that of CmLOX1, but relative levels of transcript in the various organs and tissues were similar for the two genes. Expression was highest in roots, flowers, and fruit mesocarp tissues. CmPLDα1 expression in fruit was essentially constitutive throughout development, although maximum levels occurred at 50 and 55 DAP, respectively, in middle and hypodermal mesocarp. CmLOX1 expression was generally higher in middle than in hypodermal mesocarp with maximum transcript levels occurring at 55 and 50 DAP, respectively. Overall, the patterns of expression of CmPLDα1 and CmLOX1 are consistent with a model in which their encoded enzymes act in tandem to promote or accelerate senescence in fruit mesocarp tissues.

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Fahrettin Goktepe and Harrison Hughes

`Crimson Sweet' watermelon was transformed with a copper inducible ipt gene. Clonally propagated transformed and non-transformed plants were sprayed with three different concentrations (0, 50, 100 mm) of CuSO4 at the 2–3 leaf stage twice in a 24-hour period prior to their inoculation with fusarium wilt organisms. Plants were inoculated via root dip with two different isolates of Fusariumoxysporum sp. niveum Race 2. The pathogenic strains of Fusariumoxysporum sp. niveum Race 2, Fl 99-1, and Calg 15-19, were cultured on PDA solid medium and then transferred to a sterile flask filled with 50 mL of potato dextrose broth (PDB) liquid medium. These flasks were placed on a shaker at 100 rpm for 4–5 days before the inoculation date and the final concentration of 106 spores/mL was adjusted for inoculation. Roots of the experimental units were gently washed and were then infected by dipping them in to the beaker containing the isolates for 1 min. Inoculated plants were transferred to planting trays and maintained under growth chamber conditions. Plants then were watered as needed. Fusarium wilt symptoms initially appeared approximately 7–10 days after the infection. The Cu-ipt transformants exhibited a clear and significant resistance over non-transformed plants. The severity of disease development was relatively higher in the control Cu-ipt and non-transformed plants when compared to the plants treated with CuSO4. The control Cu-ipt plants were comparatively healthier than the control non-transformed plants. Infected plants were removed from soil 2 weeks after inoculation, washed and the stems were cut vertically for rating of browning of the vascular system.