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Kathryn Kamo and Bong Hee Han

the transformation of lilies. Agrobacterium -mediated transformation of Lilium longiflorum and Oriental lilies has been demonstrated ( Hoshi et al., 2004 ; Mercuri et al., 2003 ; Ogaki et al., 2008 ). Biolistic-mediated transformation has been

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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

methods have been developed, such as electric shock ( Sagi et al., 1994 ), biolistic bombardment transformation ( Becker et al., 2000 ; Chee et al., 2005 ; Daniels et al., 2018 ; Sági et al., 1995 ; Vishnevetsky et al., 2011 ), Agrobacterium -mediated

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M. S. Strefeler

Genetic transformation of cut roses may greatly facilitate cultivar improvement programs by shortening the time required to introduce new genes into elite germplasm. The biolistic process offers a very promising method for the genetic transformation of roses.

The biolistic process uses high velocity mircoprojectiles (gold or tungsten) to carry foreign DNA into cells. This process has been shown to be useful for genetic transformation of many organisms. The first step in taking advantage of this process is to optimize the factors which affect transformation efficiency.

Several factors that have a significant affect on transformation efficiency were examined in an effort to optimize the biolistic process for gene transfer in roses. The factors examined were type of tissue (leaf segments, petioles, callus, etc), bombardment distance, the number of bombardments, DNA construct and microcarrier velocity.

The reporter gene, GUS, was used for determining transformation efficiency in this study. GUS was carried on several plasmid constructs which also contained antibiotic resistance (kanamycin or streptomycin. Efficiency of gene transfer was determined by calculating the number of transiently expressing GUS cells for each combination of factors.

Results of this study will be discussed and summarized.

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B.J. Ahn, J.S. Choi, K. Kamo, and J.W. King

Biolistic transformation methods for zoysiagrasses (Korean lawngrass) were developed and used to introduce a herbicide-resistant trait. Embryogenic calli were induced from mature caryopses on MS medium supplemented with 2 mg/L of 2,4-D, and used to establish liquid agitation cultures. The cultures have been maintained over a year without loss of the embryogenic competence. A particle bombardment method was optimized for zoysiagrass based on transient gusA gene expression. The most transient GUS expression upon bombardment treatments occurred at 1100 psi of helium pressure with 10 cm of particle flying distance and 0.125 M sorbitol preculture treatment. Promoters suitable for zoysiagrass were compared, and actin and ubiquitin promoters were found effective in expressing gusA gene. Vector DNAs containing a herbicide resistant gene (bar), pBY505, were introduced into embryogenic cells of zoysiagrass using the optimized method. Total 194 putatively transformed plants were regenerated from 60 biolistic plates for over 6 months through selection culture containing 4-10 mg/L of phosphinothricin. Regenerants were grown in potting soil in greenhouse and sprayed with 1.7 g/L of Ignite herbicide. The transgenic plants showed various levels of resistance to the herbicide, while untransformed control plants were all dead. Recombination of the bar gene into the genomes of the transformants were confirmed through PCR and Southern blot analysis.

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J.L. Anaya-López, I. Torres-Pacheco, M. González-Chavira, J.A. Garzon-Tiznado, J.L. Pons-Hernandez, R.G. Guevara-González, C.I. Muñoz-Sánchez, L. Guevara-Olvera, R.F. Rivera-Bustamante, and S. Hernández-Verdugo

Screening for resistance to mixed infections with pepper huasteco virus (PHV) and pepper golden mosaic virus (PepGMV) was carried out on plants representing wild pepper accessions collected in different states of México. One accession collected in Yucatán (BG-3821) corresponded to Capsicum chinense Jacq., and three collected from Michoacán (BG-3818), Tamaulipas (BG-3820), and Sinaloa (BG-3819) were identified as C. annuum L. Forty-eight plants were initially inoculated with a 1:1 mix of PHV and PepGMV DNAs by a biolistic method. Those plants that did not show typical symptoms after the biolistic method, were inoculated by grafting. Half of the plants (24) were highly susceptible, while the other half expressed different degrees of resistance. Of the resistant individuals, eight plants were asymptomatic and viral DNA of both viruses was detected in low levels. Two individuals showed delayed symptoms 34 days after symptom expression in the control plants. This delay was correlated with an increase in PHV DNA levels when plants became symptomatic. The remaining 14 plants showed symptom remission in newly developed leaves at 31 days postinoculation, and this asymptomatic effect was correlated diminished PHV DNA within the plants. Our results suggest that the resistance shown by some individuals to geminivirus mixed infections (PHV and PepGMV) is likely due to constrains in viral movement.

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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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Zhanyuan Zhang, A. Mitra, and D.P. Coyne

Optimization of parameters influencing biolistic transformation is a crucial stage towards repeatable transformation of common beans. However, there has been no published study on such optimization of this crop species in a helium particle delivery system (BioRad). Using an intron-containing β-glucuronidase (GUS) gene as a reporter, we optimized several critical parameters of biolistic PDS-1000/He delivery system for common bean transformation. The target explant tissues included cotyledons, zygotic embryos, and meristemic shoot tips suitable for organogenesis. Thus, pretreatment of target tissues with osmotic medium containing 0.15–0.25 m mannitol and 0.15–0.25 m sorbitol, positioning of target tissues in 4 cm microcarrier flying distance, the use of 1.6-μm gold particle and high concentration of coating DNA, and bombardment of young immature tissues twice at 2000 psi, etc., significantly increased transformation rate and achieved the best coverage and penetration of the meristemic areas involved in direct shoot organogenesis.

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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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Ana Cristina M. Brasileiro, Francisco J. Lima Aragão, Sílvia Rossi, Diva Maria A. Dusi, Leila M. Gomes Barros, and Elíbio L. Rech

To develop an efficient protocol for Agrobacterium-mediated transformation of common bean (Phaseolus vulgaris L.) and tepary bean (P. acutifolius A. Gray), we have tested the susceptibility of six genotypes to eight Agrobacterium tumefaciens and two A. rhizogenes strains. The virulence of the Agrobacterium strains was shown to be genotype dependent. In general, the tumors observed on common bean cultivars were larger than those observed on tepary bean cultivars. The A. tumefaciens AT8196 and Ach5 strains and the A. rhizogenes 8196 strain induced the best responses in all genotypes tested. Polymerase chain reaction (PCR) analysis confirmed the presence of T-DNA in tumors derived from inoculation with three A. tumefaciens strains in common beans. Apical meristems of P. vulgaris cv. Jalo were bombarded with tungsten microprojectiles and then inoculated with an A. tumefaciens wild-type strain (Ach5). One month later, the explants showed a high frequency of tumor formation (50% to 70%). Similarly, when bombarded meristems were inoculated with an A. tumefaciens disarmed strain (LBA4404/p35SGUSINT), 44% of them showed substantial sectors of GUS activity, suggesting the expression of introduced gene. The bombardment/Agrobacterium system appears to be a promising method to stably transform bean through the regeneration of plants directly from transformed apical meristems.

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Alejandrina Robledo-Paz, José Luis Cabrera-Ponce, Víctor Manuel Villalobos-Arámbula, Luis Herrera-Estrella, and Alba Estela Jofre-Garfias

Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Embryogenic calluses, derived from garlic cultivar `GT96-1', were bombarded with plasmid DNA containing genes coding for hygromycin phosphotransferase and β-glucuronidase. Putatively transformed calluses were identified in the bombarded tissue after 4 months of selection on 20 mg·L-1 hygromycin B. The transgenic nature of the selected material was demonstrated by GUS histochemical assay and Southern blot hybridization analysis, and twenty transgenic plants were regenerated.