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Moon Kyung Ko, Hyunchul Soh, Kyung-Moon Kim, Young Soon Kim, and Kyunghoan Im

Agrobacterium tumefaciens . The plasmid vector pCAMBIA1301 (CAMBIA, Clayton Australia), which contains β-glucuronidase ( uid A) and hygromycin phosphotransferase ( hpt ) genes under the control of the CaMV35S promoter, was transferred into A. tumefaciens

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Fengge Hao, Lirong Wang, Ke Cao, Xinwei Wang, Weichao Fang, Gengrui Zhu, and Changwen Chen

, 2004 ; van Loon et al., 1998 ). Agrobacterium tumefaciens , a soil-borne bacterium, causes formation of crown galls in many plant species. During infection, a specific segment of the tumor-inducing plasmid, the transfer DNA (T-DNA), is transferred from

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Xiaojuan Zong, Brandon J. Denler, Gharbia H. Danial, Yongjian Chang, and Guo-qing Song

-Cardenas, M.L. 2013 Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato ( Solanum lycopersicum L.) cultivar Micro-Tom Plant Cell Rep. 32 239 247 Dai, W.H. Magnusson, V. Johnson, C. 2007 Agrobacterium -mediated

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Keun H. Cho, Joo Young Kim, Maria I. Alvarez, Veronica Y. Laux, Lauren K. Valad, Joshua M. Tester, Thomas A. Colquhoun, and David G. Clark

novel GFP-like fluorescent genes. Therefore, seven fluorescent genes ( DsRed2 , E2Crimson , TurboRFP , ZsGreen1 , ZsYellow1 , rpulFKz1 , or aeCP597 ) were introduced into petunia plants by Agrobacterium tumefaciens –mediated transformation to

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Christine Berry, J. Van Eck, S. Kitto, and A. Smigocki

Orangemint leaf disks were infected with three strains of Agrobacterium tumefaciens: A281, a hyper-virulent strain containing plasmid pTiBo542; C58, a strain containing nopaline Ti plasmid pTiC58; and A136, a derivative of C58 lacking the nopaline Ti plasmid. After a 24 or 48 hr cocultivation, leaf disks were placed on a medium containing MS salts and vitamins, 2% sucrose, and 200 μgm l-1 cefotaxime. Callus formed only on those leaf disks infected with A281. Five callus lines (R-12, -71, -73, -81, -83) were putatively transformed based on succinopine production. Definitive transformations were confirmed via DNA slot blot analysis. All callus lines assayed (R-11. -12, -41, -73, -83) hybridized to a 7.7 KB fragment from the T-DNA region.

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Zoran Jeknic, Stephen P. Lee, Joel Davis, Richard C. Ernst, and Tony H.H. Chen

Experiment Station technical paper no. 11467. We thank Barbara M. Reed and William M. Proebsting for their helpful reviews. Agrobacterium tumefaciens LBA4404(pTOK233) used in this project was obtained through a research agreement with Japan Tobacco, Inc

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J.M. Al-Khayri, F.H. Huang, T.E. Morelock, and H.T. Zhang

The objective of this study was to determine the efficacy of Agrobacterium tumefaciens in transforming spinach (Spinacia oleracea L.) callus. Callus was induced from leaf disks of `Baker' on Murashige and Skoog (MS) medium supplemented with 2 mg L-1 kinetin and 0.5 mg L-1 2,4-D. Callus was cut into 2-mm pieces, and 0.5 g of callus was placed in each 250-ml flask which contained 20 ml of MS liquid medium. The suspension cultures were inoculated with 100 μl of an overnight culture of A. tumefaciens harboring pMON 9749 (provided by S. Rogers, Monsanto Co., St. Louis), a plasmid cointegrated with kanamycin resistance and β -glucuronidase (GUS) genes. After coculturing for 2 days at 22C with shaking at 100 rpm, the medium was replaced with selection medium containing (in μg/ml) 75 kanamycin, 100 cefotaxime, and 200 carbenicillin and maintained for 3 weeks. Transient expression of GUS gene in transformed cells was detected with X-glu assay. This method resulted in a high level of transformation and provides the first report of transformation in spinach. This study was funded by a grant (92-B-32) from the Arkansas Science & Technology Authority.

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F.A. Hammerschlag, R.H. Zimmerman, and A.C. Smigocki

`McIntosh' apple shoots were inoculated in vitro with Agrobacterium tumefaciens strain tms328::Tn5 (tms) carrying a functional cytokinin gene. Callus tissue, removed from the infected stems, produced shoots on shoot proliferation medium. After three subcultures, axillary shoot production from a tms-infected putative transformant was eight times that of controls. Subsequent shoot production on three different levels of BA (3, 6 and 10 uM) was significantly greater than from controls on all levels of BA. PCR analysis of putative transformants revealed an expected 503 bp DNA fragment corresponding to the amplified portion of the cytokinin gene. After 6 months of in vitro propagation, proliferation rates of shoots obtained from the original transformants were similar to the controls and the expected PCR fragment of 503 bp could only be detected by Southern analysis. Even though the T-DNA appears to be lost from the apple genome, the data suggest that the tms strain may be useful in co-infection experiments to induce shoot formation, thus avoiding difficult regeneration procedures.

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James R. McKenna and Lynn Epstein

Crown gall, caused by the common soil-borne bacterium Agrobacterium tumefaciens, can be an economic problem in walnut nurseries and production orchards in California. The principal rootstocks used for commercial walnut production in California are the native Northern California black walnut, Juglans hindsii, and “Paradox,” which are interspecific hybrids between a black walnut, primarily J. hindsii, as the maternal parent, and J. regia, the English walnut, as the paternal parent. Recent evidence has shown that some commercial black walnut trees producing Paradox hybrid seedlings are actually hybrids between J. hindsii and two other North American black walnut species, J. major and J. nigra. Here, we document that there was a higher incidence of crown gall on Paradox (J. hindsii ×J. regia) than on J. hindsii in three sites with natural soil inoculum. Paradox seedlings (with a female parent that was primarily J. hindsii with some J. nigra) inoculated with A. tumefaciens on the roots during transplanting had a higher incidence of crown gall than either J. hindsii or J. regia. When stems were inoculated with A. tumefaciens, J. hindsii ×J. regia populations had significantly larger galls than either J. hindsii or J. regia. Similarly, in stem inoculations on four out of six Paradox genotypes with a hybrid black walnut maternal parent, the progeny produced significantly larger galls than either J. hindsii or J. regia. However, two Paradox populations from black walnut hybrids that contained J. major, J. nigra, and J. hindsii produced galls that were no different in size than in the black walnut species and J. regia. Results suggest that J. regia and black walnut species are less susceptible to crown gall than most Paradox populations.

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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.