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Sue A. Hammar and Rebecca Grumet

We sought to develop efficient regeneratio nand transformation procedures for cucumber. Factors tested for regeneration included: hormone types and levels, genotype, explant source, and environmental conditions. Optimum regeneration was obtained using cotyledon pieces from 4 day old GY14A seedlings and culturing for 3 weeks under cool white lights (30-40 uE-2 s -1) on MS medium supplemented with 1.0 mg/l 2,4-D, 0.5 mg/l BA, 0.3 mg/l ABA, 30 g/l sucrose, 1 g/l MES, and 3.07 g/l Scott gelrite. Shoots developed via somatic embryogenesis ca. 2 wk after explants were transferred to MS supplemented with 20 g/l sucrose, 1 g/l MES, and 4.37 g/l gelrite. Ca. 80% of the explants produce shoots, 1/3-1/2 of which produce rooted plantlets; total time from explant to rooted plantlet is ca. 8 wks. Transformation experiments utilized Agrobacterium tumefaciens strains LBA4404 bearing the CIBA-GEIGY pCIB10 vector with a selectable marker gene for kanamycin resistance. Optimal conditions include 45 mg/l kan, 10 min inoculation and 3 day co-cultivation. Preliminary evidence suggests that tobacco nurse cultures increase transformation efficiency. Transgenic plants were confirmed by Southern or dot blot analysis.

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Van Quy Le, Serge Overney, Binh Nguyen Quoc, and Serge Yelle

Tomato is one of the most important crop species where the introduction of foreign genes is expected to have a major impact on agriculture. Several transformation methods exist that rely on the cocultivation of various tissue or organ explants. However, tomato is still considered more difficult to transform than species such as Petunia hybrida and Nicotiana tabacum and can show widely varying success rates. Using cotyledonous explants, we propose a highly efficient procedure of Agrobacterium-mediated transformation and regeneration of an agricultural cultivated tomato (L. esculentum cv. Summerset). Results showed that up to 90% of the cotyledons generated callus within 3 weeks (1 to 5 calli/cotyledon) and 50% of them regenerated shoots in another 3 weeks. Finally, it resulted in 50 to 100 independent transgenic plants per 100 inoculated explants within 10 weeks. These results are at least 40% more efficient than those of already published protocols. Moreover, up to 95% of the regenerated plants that form vigorous de novo roots under the antibiotic selection tested positive for the GUS assay. Screening by PCR for the presence of the T-DNA genes gave the predicted DNA fragment bands. This high efficiency procedure was mainly achieved by 1) an adequate optimization of the hormone composition and concentration of the successive culture media; 2) the fresh explant wounding before the Agrobacterium infection (important for optimal cell transformations); 3) the explant position, inside down for callus induction and coculture period, and upside down for the selection and organogenesis period (important for antibiotic selection).

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Sue A. Hammar and Rebecca Grumet

We sought to develop efficient regeneratio nand transformation procedures for cucumber. Factors tested for regeneration included: hormone types and levels, genotype, explant source, and environmental conditions. Optimum regeneration was obtained using cotyledon pieces from 4 day old GY14A seedlings and culturing for 3 weeks under cool white lights (30-40 uE-2 s -1) on MS medium supplemented with 1.0 mg/l 2,4-D, 0.5 mg/l BA, 0.3 mg/l ABA, 30 g/l sucrose, 1 g/l MES, and 3.07 g/l Scott gelrite. Shoots developed via somatic embryogenesis ca. 2 wk after explants were transferred to MS supplemented with 20 g/l sucrose, 1 g/l MES, and 4.37 g/l gelrite. Ca. 80% of the explants produce shoots, 1/3-1/2 of which produce rooted plantlets; total time from explant to rooted plantlet is ca. 8 wks. Transformation experiments utilized Agrobacterium tumefaciens strains LBA4404 bearing the CIBA-GEIGY pCIB10 vector with a selectable marker gene for kanamycin resistance. Optimal conditions include 45 mg/l kan, 10 min inoculation and 3 day co-cultivation. Preliminary evidence suggests that tobacco nurse cultures increase transformation efficiency. Transgenic plants were confirmed by Southern or dot blot analysis.

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Salvador Guzmán-González, Pedro Valadez-Ramírez, Rosa-Edith Robles-Berber, Laura Silva-Rosales, and José-Luis Cabrera-Ponce

Biolistic genetic transformation of plants with viral genes is a method for controlling plant virus diseases; however, optimization of the particle bombardment parameters according to the transformation system is a key factor for an appropiate transgene expression and, therefore, a stronger resistance mechanism in transgenic plants. In order to optimize biolistic parameters, somatic papaya (Carica papaya L.) cv. Maradol embryo masses were bombarded with the CAMBIA 1301 plasmid construction that contains the coat protein gene (CP) of the papaya ringspot virus isolate of Colima, Mexico, driven by the double constitutively CaMV 35S promoter and flanked for the GUS and hygromycin (hpt) resistance genes. Particle bombardment protocol was carried out using the Helios™ Gene Gun device (BioRad) and the manufacturer's instruction manual. Helium pressure (50, 100, and 150 psi) and gold particle size (0.6, 1.0, and 1.6 μm) were evaluated. Five days after bombardment, somatic embryo clusters were used for GUS transient expression and, during 2 months, were selected into 50, 75, and 150 mg·L-1 hygromycin-containing media to its later CP-PCR detection. Results showed that 50 psi and 1.0 μm were the two optimal values for the assayed analyses. This is the first report of genetic transformation of papaya using the Helios™ Gene Gun device as a new tool compared to conventional PDS-1000/He.

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Mark E. Lewis and Fred A. Bliss

Ten common bean (Phaseolus vulgaris L.) lines—including cultivars, breeding lines, and one wild line—were evaluated for susceptibility to Agrobacterium tumefaciens strain C58 by stab-inoculating intact shoot tips of germinating seeds. Significant differences for tumor frequency and size were found on the resulting 3-week-old seedlings. UW 325, a wild bean, had the highest rate of tumorigenesis; `Olathe', a dry bean cultivar, had the lowest. Uninoculated excised shoot tips cultured in media with BA or BA plus NAA exhibited differences in phytohormone sensitivity, as evidenced by callusing and root initiation. The cultivar Montcalm seemed to be highly sensitive, while `Olathe' was relatively insensitive. Fluorometric GUS assays of shoot tips from germinating seeds inoculated with the disarmed GUS-containing A. tumefaciens strain C58C1(pGV3850/pKIWI105) showed that UW 325 had the highest level of GUS activity. `Montcalm' had a high rate of tumorigenesis but a low level of GUS activity; this anomaly was attributed to its high phytohormone sensitivity. The use of the virulence-inducing compound acetosyringone in the inoculum culture medium did not alter genotypic differences (ranks) in susceptibility. Histochemical GUS assays of inoculated UW 325 shoot tips showed that 60% of the apexes exhibited one or more transformation events. Chemical names used: β-glucuronidase (GUS); α-naphthaleneacetic acid (NAA); N-(phenylmethyl)-1H-purin-6-amine (BA).

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Jeung-Sul Han* and Chang Kil Kim

A procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 carrying a binary vector pCAMBIA3301, which contains glufosinate ammonium-resistant (bar) and the reporter (gus) genes, is describe. Infection was the most effective (highest infection frequency and index) when explants were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.001-0.1 mg/L L-a-(2-aminoethoxyvinyl) glycine (AVG). Transgenic plants were obtained with frequencies of about 0.2% when the explants were cultured on selection medium (MS medium supplemented with 3.0 mg/L BAP, 0.5 mg/L AgNO3, 500 mg/L cefotaxime, 2.0 mg/L DL-phosphinothricin, 0.3% sucrose and 0.8% Plant Agar. A histochemical gus assay, PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progenies showed that the transgenes were inherited in a Mendelian fashion. To our knowlege, this study represents the first report for Agrobacterium-mediated transformation in bottle gourd, rootstock for watermelon and other cucurbit crops in many countries.

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Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson

Plant regeneration of ginseng has been known to be difficult, and there are a few reports on plant regeneration of ginseng via somatic embryogenesis. In vitro flowering has, however, been one of the major drawbacks in these regeneration systems in which BA and GA3 were included in germination and shoot multiplication media. Multiplication of adventitious shoots from a single somatic embryo, abnormal morphology, and vitrified shoots were also observed. All these facts have made successful acclimatization of ginseng plantlets difficult. The purposes of this study were 1) to establish the plant regeneration system via organogenesis, 2) to improve normal plant regeneration via somatic embryogenesis, 3) to improve the efficiency of plant regeneration from protoplast culture, 4) to understand the acclimatization process, 5) to develop effective genetic transformation protocol. Data in relation with all these studies are presented in detail.

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Yali Liu, Fuling Hao, Rui Meng, and Weirong Xu

The enzyme ACC oxidase (ACO), encoded by a small multigene family in many plants, catalyzes the terminal step in the ethylene biosynthesis pathway. In this research, based on the total RNA isolated from the flowers of Asia hybrids `Pollyanna' and Oriental hybrids `Sorbonne', we obtained two cDNA fragments of ACO genes (Genbank accession DQ062133 and DQ062134) by RT-PCR technique. The two cDNA fragments were reversely inserted into plant expression vector pWR306 respectively, and constructed two antisense ACO gene expression binary vectors harboring hygromycin phosphotransferase (hptII), glucuronidase (uid A), and a green fluorescent protein (GFP) gene in the T-DNA region. We have developed a system to produce transgenic plants in LiLium via Agrobacterium tumefaciens-mediated transformation of calli. Transformants were subjected to GFP expression analysis, PCR assay, and Southern hybridization to confirm gene integration.

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A. Virginia Freire, David A. Lightfoot, and John E. Preece

Efficient genetic transformation could enhance coffee breeding, which is limited by its long generation time and narrow genetic base. Three explant types of three coffee cultivars were inoculated with 14 strains of Agrobacterium spp. Callus and hairy roots were produced with 13 of the 14 strains tested. With A. tumefaciens, nopaline strains were more effective than octopine strains. Cucumopine and mannopine strains of A. rhizogenes were both effective in inducing hairy roots and callus. PCR amplification of a 0.72 Kb fragment of T-DNA encoding a portion of the ipt gene was achieved with DNA from A. tumefaciens strain A208 and with putatively transformed tissue inoculated with A208. No amplification was observed with virB in putatively transformed tissue which indicates it was not contaminated with Agrobacterium. We conclude that coffee can be genetically transformed by some Agrobacterium strains.

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Charleen M. Baker and William E. Dyer

Our goal was to develop efficient regeneration protocols for safflower that could be used in conjunction with Agrobacterium tumefaciens -mediated transformation to introduce genes conferring economically important traits. Direct regeneration of whole plants has been achieved from cotyledon and hypocotyl explants of 30-day-old `Centennial' and `Montola' seedlings. Explants transformed with Ti plasmids containing NPTII and the β-glucuronidase (GUS) reporter gene produced kanamycin-resistant calli and shoots testing positive for GUS activity. Current work is incorporating the bar gene into appropriate Ti plasmids that will be used to confer glufosinate herbicide resistance to elite safflower cultivars. An esterase gene from Bacillus subtilis will be introduced to confer resistance to Alternaria carthami leaf spot disease.