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.
Salvador Guzmán-González, Pedro Valadez-Ramírez, Rosa-Edith Robles-Berber, Laura Silva-Rosales, and José-Luis Cabrera-Ponce
Kathryn Kamo and Bong Hee Han
particles. Gold particles were coated with plasmid DNA according to the method of Sanford et al. (1993) . The gene gun had a 1-cm gap and 1-cm flying membrane distance. The target distance was either 9 or 12 cm, and cells were bombarded at 900, 1100, or
Xiaojian Ye, Susan K. Brown, Ralph Scorza, John Cordts, and John C. Sanford
Physical and biological parameters affecting the efficiency of biolistic transformation of peach were optimized using ß-glucuronidase (GUS) as a reporter gene, such that efficiency of transient GUS expression in peach embryo-derived callus was increased markedly. Transient expression was also obtained in embryonic axes, immature embryos, cotyledons, shoot tips, and leaves of peach. Stable expression of a fusion gene combining neomycin phosphotransferase (NPTII) and ß-glucuronidase activities has been achieved in peach embryo calli. Sixty-five kanamycin-resistant callus lines were obtained from 114 pieces of bombarded calli after 4 months of selection. Nineteen of the 65 putative transformant lines produced shoot-like structures. Seven lines were examined to confirm stable transformation using the colorimetric GUS assay and PCR analysis. All seven lines showed GUS activity. PCR analysis confirmed that, in most of the putative transformants, the chimeric GUS/NPTII gene had been incorporated into the peach genome. The transgenic callus lines were very weakly morphogenic, presumably because the callus was 5 years old and no transgenic shoots developed from this callus. Results of this research demonstrate the feasibility of obtaining stable transgenic peach tissue by biolistic transformation.
Carol Gonsalves, Baodi Xue, Marcela Yepes, Marc Fuchs, Kaishu Ling, Shigetou Namba, Paula Chee, Jerry L. Slightom, and Dennis Gonsalves
A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.
Zhanyuan Zhang, D.P. Coyne, and A. Mitra
Gene transfer can provide plants with a novel source of disease resistance. Two different antibacterial peptides, Shiva-1 and lactoferrin, were tested in vitro for antibacterial activity. The former is from cecropin B in insects, and the latter from human or mammal fluids such as milk. Both peptides exhibited high antibacterial activity against all tested gram-negative phytopathogenic bacterial strains. Lactoferrin was more lethal than Shiva-1. A particular lactoferrin domain showed a much higher activity against bacterial strains. A gene encoding lactoferrin was then transferred to Nicotinia tabacum L. xanthi-nc to evaluate the gene expression using Agrobacterium. Stable transformation was confirmed by Southern, Northern, and Western blot analysis. Delayed wilting of the transgenic plants inoculated with Pseudomonas solanacearum was observed. A significant positive relationship between the gene expression levels and resistance was also found by either Northern or Western blotting. Biolistic transformation using a gene gun is currently underway to transfer this novel gene to common beans.
ASHS 2007 Presidential Address: William R. Woodson
William R. Woodson
culture led the way for genetic modification of plants. Horticultural science was at the center of plant biotechnology development of the gene gun, when John Sanford blasted away at plant cells in his laboratory at Cornell. This work was critical to the
Hiroaki Ito, Masaki Ochiai, Hiroaki Kato, Katsuhiro Shiratake, Daigo Takemoto, Shungo Otagaki, and Shogo Matsumoto
particles coated with total RNSs at 1650 psi pressure using the PDS-1000/HeTM Particle Delivery System (Bio-Rad Laboratory, Inc.) or 80–100 psi pressure using the Helios Gene Gun System (Bio-Rad Laboratory, Inc.). After particle bombardment, the plants
Christopher C. Dickinson, Alexandra J. Weisberg, and John G. Jelesko
-mediated transformation proves problematic. Biolistic transformation is the acceleration of nucleic acid–coated microparticles to high velocity (using a so-called gene gun) resulting in entry into the plant cell cytoplasm, where the nucleic acid subsequently uncoats from
Giseiry Rosa-Valentín, Linda Wessel-Beaver, and Jose Carlos V. Rodrigues
controlled by a single recessive gene. Guner (2004) tested 1644 cultigens of watermelon using ZYMV-FL. The accessions with the highest resistance to ZYMV were PI 386016, PI 386019, PI 485580, PI 494529, PI 537277, PI 560016, PI 595200, and PI 595203. Guner
Hongmei Ma, Margaret Pooler, and Robert Griesbach
backbone region of the plasmid vector identical to the S-R construct, as described in Ma et al. (2008) . Biolistic transformation was carried out using a gene gun, and transient expression was monitored using fluorescence microscopy ( Ma et al., 2008