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Tao Dong, Fang-cheng Bi, Yong-hong Huang, Wei-di He, Gui-ming Deng, Hui-jun Gao, Ou Sheng, Chun-yu Li, Qiao-song Yang, Gan-jun Yi, and Chun-hua Hu

grew slowly and gradually browned and died, whereas transformed cell clusters continuously proliferated. The first, second, and third generations of the screened embryogenic cell suspensions were stained with GUS. The results showed that the positive

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Patricia Yolanda Zapata-Castillo, Adriana-Canto Flick, Guadalupe López-Puc, Anabel Solís-Ruiz, Felipe Barahona-Pérez, Nancy Santana-Buzzy, and Lourdes Iglesias-Andreu

zygotic embryos ( Buyukalaca and Mavituna, 1996 ), and from leaves ( Kintzios et al., 2001 ). With the exception of Buyukalaca and Mavituna (1996) , who obtained somatic embryos of C. annuum from cell suspensions, in all these reports, the somatic

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Jing-Tian Ling and Roger J. Sauve

Leaf segments of greenhouse-grown Ulmus americana L. plants cultured on a Murashige and Skoog basal salts medium supplemented with 0.22 mg/L thidiazuron formed friable type of callus and regenerated shoots. This friable callus readily formed a cell suspension when the callus was placed in a liquid MS medium containing 2 mg/L 1-naphthaleneacetic acid and 1 mg/L 6-benzylaminopurine. Shoots were regenerated from 3-month-old suspension cell cultures after the suspension cells had been cultured on solid medium. Shoots developed roots on MS medium containing 0.1 mg/L indole-3-butyric acid. Intact plants were successfully established in soil.

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Huiling Wang, Wei Wang, Weidong Huang, and Haiying Xu

inhibit the activity of benzoic acid hydroxylase enzyme in the SA biosynthetic pathway ( León et al., 1995 ), so it can be used as an effective candidate inhibitor for the synthesis of SA from phenylalanine ( Liu et al., 2006 ). Plant cell suspension

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W. Bundithya and S.L. Kitto

Thlaspi caerulescens (Brassicaceae), known as a Zn hyper accumulator, is able to accumulate and tolerate Zn, Ni, Cu, and Cd at high concentrations in its biomass. We are examining the feasibility of using cell suspensions of T. caerulescens and B. napus to study the effect of selected heavy metals on growth and nutrient uptake. Callus was initiated by culturing seedlings on basal medium containing MS salts supplemented with MS or B5 vitamins, 1, 2, 5, or 10 mg 2,4-D/liter, and 0.7% Phytagar. Cell suspensions were initiated by transferring calli to liquid basal medium containing MS or B5 vitamins, and 1 or 2 mg 2,4-D/liter, and were incubated on a gyratory shaker at 120 rpm. Growth of suspensions inoculated at 0.2, 0.4, or 0.6 g/25 ml was monitored for 13 days. Optimal conditions required to initiate and maintain suspension cultures of T. caerulescens and B. napus include MS medium supplemented with B5 vitamins and 1 mg 2,4-D/liter, an inoculation density of 0.4 g/25 ml, and a 2-week subculture schedule.

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Julie A. Russell, Mihir K. Roy, and John C. Sanford

The biolistics process uses high velocity microprojectiles to carry foreign DNA into cells. Though biolistics has already proven to be useful fo r a wide range of species, improvements are still needed, and many of the factors which affect transformation efficiency have not been defined. In our experiments, cell suspensions of Nicotiana tabacum (NT1 line) were used as a model to identify these factors. The most critical factors for high efficiency transformation were: cell age, microprojectile type & size, DNA construct, osmoticum in the bombardment medium, use of a new helium-driven biolistic device, and the handling and growth environment of the cells after bombardment. By optimizing these factors, an average of 7,000 transiently expressing GUS cells and 800 kanamycin resistant colonies were obtained per bombarded plate. The high efficiency and rapid results (2 days transient/4 weeks stable) of the NT1 model system make it useful for cell biology studies and for testing DNA constructs.

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Weenun Bundithya and Sherry L. Kitto

Thlaspi caerulescens (Brassicaceae), known as a Zn hyperaccumulator, is able to accumulate and tolerate Zn at high concentrations in its biomass. Cell suspension cultures of Thlaspi caerulescens J et C Presl and B. napus `Westar' have been initiated to study the effect of high Zn concentrations on growth and nutrient uptake. Preliminary studies determined the optimal conditions for subculturing and maintaining cultures. Cell suspensions grew best on Murashige and Skoog medium supplemented with B5 vitamins and 1 mg 2,4-D/liter at 0.4 g/25 ml inoculation density, and with a 2-week subculture period. In an initial experiment, cell suspensions were cultured in media containing 1.96 ppm Zn (basal) or 49 ppm Zn (25x). Media and tissue samples were collected at days 0, 4, 7, 10, and 13, and their nutrient content was analyzed by ICP-AES. Thlaspi and Brassica cell suspensions grew equally well on both media. For both species, uptake patterns of Ca, K, Mg, Mn, and P were not significantly different between the two media; however, >97% of the P was taken up within 2 weeks. Zinc concentration was reduced during the first 4 days (lag phase) in the high-Zn medium, with 27% and 41% taken up by the Thlaspi and Brassica cultures, respectively. Thlaspi took up significantly less Zn than did Brassica. By day 13, Thlaspi and Brassica tissue collected from the high-Zn medium contained 10x and 32x, respectively, more Zn when compared to tissue grown on basal medium.

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Violeta Colova-Tsolova, Rachel Gollop, Sharon Farchie, Sylvie Even, Nahman Shahar, and Avi Perl

Embryogenic cell suspension was developed from in vivo anthers of seedless grape cv. Sugarone. Agrobacterium genetic co-transformation was realized with two vectors carried respectively two different reporter genes: hpt and nptII, and three (1+2) agronomicaly beneficial genes encoding for proteins that are involved in fungus disease resistance. The effciency of transformation procedure and integraton of foreign genes was verified by hystochemical assay as a first step after insertion in embryogenic suspension two different constructs with Gus-reporter gene under control of different promotors. PCR assay and Southern blot analysis were used to confirm the co-transformation in regenerated grape plants.

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F. Mark Schiavone and M.E. Wisniewski

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Yan Ma, M. Nurul Islam-Faridi, Charles F. Crane, David M. Stelly, H. James Price, and David H. Byrne

To our knowledge, there has been no published technique to produce consistently high-quality slides of somatic chromosomes of roses (Rosa sp.). Therefore, various pretreatments, fixatives, digestions, stains, and maceration and squashing methods were tested to identify a procedure to produce clear, well-spread chromosomes from shoot tips. The best results were obtained after pretreatment in a mixture of 0.1% colchicine and 0.001 m 8-hydroxyquinoline for 4 h, and fixation in 2 acetone: 1 acetic acid (v/v) with 2% (w/v) polyvinylpyrrolidone. The darkest-stained chromosomes were obtained with carbol-fuchsin staining of air-dried cell suspensions that had been spread in 3 ethanol: 1 acetic acid (v/v).