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Kalyani Dias, Suzanne M.D. Rogers, and Ronald J. Newton

Citrus is one of the major horticultural cash crops in the Southern United States. Several attempts have been made to genetically improve present citrus cultivars using biotechnology. We report transformation of citrus (Citrus sinensis (L.) Osbeck `Hamlin') suspension cultures using microprojectile bombardment.

A thin layer of seven day-old suspension cultures, grown in HH medium, were transferred to Whatman #1 filter paper at a cell density of 0.15 mg/ml fresh weight. The cells were bombarded with 1.112μM diameter, DNA-coated, tungsten particles using the Dupont PDS 1000 biolistic system. Plasmid PRT 99 GUS containing the marker genes β-Glucuronidase (GUS) and NPT II, with a 35S promoter, and NPT II terminators was used in transformation.

GUS activity was monitored on a time scale. Expression of GUS was observed after 48 hours of bombardment. Further studies are being done to enhance the transformation efficiency.

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Jane E. Knapp and Mark H. Brand

Horticultural improvements in Rhododendron require long periods of time to produce flowering plants by traditional breeding methods. In addition, new trait development by conventional genetics is limited to existing germplasm. Genetic engineering approaches to horticultural improvement offer the possibility for introduction of new traits using foreign DNA from any source. To this end, we have developed a system for the genetic transformation of Rhododendron based on microprojectile bombardment. Leaves from in vitro-grown plantlets of R. `Catawbiense Album' L. were bombarded with the marker genes uidA (GUS) in combination with nptII or hph. Two days post-bombardment, explants were transferred to shoot iniation medium containing either 50 mg/L kanamycin or 2.5 mg/L hygromycin. After 4 weeks, proliferating tissues were transferred to media containing increased levels of selective agent (100 mg/L kanamycin or 5 mg/L hygromycin, respectively). Shoots that regenerated were then excised from necrotic tissues and transferred to shoot proliferation medium containing the high level of selective agent. PCR analysis of putative transformants revealed the presence of the transgenes. Southern blot hybridization confirmed stable transgene integration. Histochemical GUS assays of transformed tissues indicated uniform expression throughout the transgenic plant. With the development of an efficient transformation system, the introduction of genes to confer useful horticultural traits becomes feasible.

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Maritza I. Tapia, P.E. Read, H.F. Kaeppler, and P.L. Herman

Direct DNA delivery via microprojectile bombardment has been successfully used to transform a wide range of species. Transformation using this system is dependent on the optimization of several parameters. These parameters involve the explant, the gene construct, and parameters in the bombardment system. DNA was delivered into bisected axillary buds of grape hybrids `Chancellor' and `Valiant'. Target tissues were bombarded with gold microprojectiles coated with GUS::NPTII fusion gene construct(pBI426). Several experiments with varying parameters were conducted in order to increase the frequency of DNA delivery. Data were analyzed as a completely random design with 6 single petri dish as a replication and 50-60 bisected axillary buds per replication in each treatment. The treatment design was the single-factor method. Higher frequencies of transient transformation were obtained using microprojectiles of 1.6 μm diameter, adding 0.15 m mannitol and 0.15 m sorbitol, under a pressure of 68.6 cm Hg and a target distance of 6 cm. After 40 days on the selection medium containing 50 mg kanamycin/L regenerated plantlets were obtained and 40% of them expressed the GUS gene. The biolistic approach using bisected axillary buds as target tissue could be a method to achieve stable transformation and transgenic grape plants.

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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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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.

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Rodney Serres, Elden Stang, Dennis McCabe, David Russell, Daniel Mahr, and Brent McCown

Genetic transformation of the American cranberry, Vaccinium macrocarpon Ait., was accomplished using electric discharge particle acceleration. Plasmid DNA containing the genes GUS (β-glucuronidase), NPTII (neomycin phosphotransferase II), and BT (Bacillus thuringiensis subsp. kurstaki crystal protein) was introduced into stem sections, derived from in vitro cultures, that had been induced to form adventitious buds. The stage of development of these adventitious buds was critical for efficient initial expression. After exposure to electric discharge particle acceleration, stem sections were cultured on a solid-phase bud-inducing medium containing 300 mg kanamycin/liter. In addition, a thin overlay of 300 mg kanamycin/liter in water was added to inhibit growth of nontransformed cells. Within 7 weeks, green shoots emerged amidst kanamycin-inhibited tissue. No escape (nontransformed) shoots were recovered, and 90% of the transformed shoots were shown through PCR and Southern blot analysis to contain all three introduced genes. GUS expression varied markedly among various transformed plants. Preliminary bioassays for efficacy of the BT gene against the feeding of an economically important lepidopteran cranberry pest have shown no consistently effective control. Potential problems with the expression of the BT and GUS genes are discussed

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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.

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Lius Suwenza and Richard Manshardt

Nine transgenic papaya clones, produced previously by microprojectile bombardment, are being characterized for frequency of somaclonal variation. Five clones have proven to be hermaphrodite. Four of these appear to have normal fertility, while the fifth has drastically reduced pollen fertility, averaging about 15% stainability with acetocarmine. Four other clones are pistillate and appear to have normal fertility, with one exception which has been demonstrated to be tetraploid (2n=36 chromosomes). One of twelve plants in a pistillate clone was a somaclonal mutant showing altered leaf and flower morphology. The transgenic clones and their sexual progenies are also being evaluated at the molecular level for expression and segregation of npt, gus, and the coat protein (CP) of papaya ringspot virus (PRV), as well as for PRV resistance.

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C.S. Prakash, U. Varadarajan, and A. S. Kumar

Development of a gene transfer system will enable rapid introduction of agronomically useful genes into elite cultivars of sweet potato. We compared microprojectile bombardment and Agrobacterium cocultivation approaches to introduce foreign genes into the genome of two sweet potato cultivars. Chimeric marker genes (gusA and kan) were successfully introduced into cvs. Jewel and TIS-70357 using both approaches. However, transgenic plants were generated in vitro using only the Agrobacterium approach. Callus and root isolates with stable expression of gusA gene were obtained using the microprojectile method. Expression of the screenable marker gusA gene was detected by histochemical assays. Integration of the introduced gene into the genome of sweet potato was confirmed by polymerase chain reaction (PCR) amplification of the kan gene and Southern blot analyses. Transgenic sweet potato plants from two cultivars are being raised and studied for quantitative expression and localization of the introduced genes. These results show that foreign genes can be successfully introduced and expressed in sweet potato. Current efforts are directed at optimizing several variables to increase the transformation efficiencies and to generate transgenic cultivars with foreign genes of agricultural importance.

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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.