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Rose E. Palumbo and Richard E. Veilleux*

A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. Hemizygous primary transformants (To) were produced from three leaf discs: 17 diploid plants from one leaf disc, three and nine tetraploids from the other two leaf discs. GFP expression was observed qualitatively under fluorescence microscopes and quantitatively with a GFP meter. Segregation ratios for tetraploid T1 seedlings fit models for single duplex insertions (35 transgenic: 1 non) or double simplex insertions (15 transgenic: 1 non). Diploid T1 seedlings segregated for deleterious traits: dwarfed size and curled leaves, as well as the GFP transgene. Similar segregation patterns in diploid families implied that all diploids may have been from the same transformation event. The cumulative segregation showed the dwarfed and curled plants fit a single recessive gene ratio (3 normal: 1 mutant), and GFP fit a double-copy insertion ratio (15 transgenic: 1 non). Six T1 selections were free of deleterious traits, consistently high expressers of GFP, and produced fertile pollen.

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Chunxian Chen, Qifa Zheng, Xu Xiang, Jaya R. Soneji, Shu Huang, Young A Choi, Madhugiri Nageswara Rao, and Fred G. Gmitter Jr.

removal of the plantlet if an early stage of screening is required. Since the green fluorescent protein (GFP) gene ( gfp ) was cloned and used as a visual marker for gene expression study ( Chalfie et al., 1994 ), it has been applied in many organisms

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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

still in progress on this topic ( Gandía-Herrero et al., 2005 ). Since the green fluorescent protein (GFP) was isolated and cloned from a jellyfish ( Aequorea victoria ), it has become an indispensable molecular marker, and several modified versions have

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Xiaobo Sun, Yanming Deng, Lijian Liang, Xinping Jia, Zheng Xiao, and Jiale Su

combined with green fluorescent protein of pX-DG-vector ( Chen et al., 2009 ) to yield a fusion protein GFP-SbPIP1. Next, the fusion expression vector pX-DG-SbPIP1 was introduced into onion epidermal cells using a gene gun (PDS-1000; Bio-Rad, Hercules, CA

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Ruining Li, Wenwen Huang, Xiaoxiao Wang, Xiaoying Liu, and Zhigang Xu

of potato plantlets in vitro than green. LEDs are widely used in plant growth chambers and are useful in plant tissue culture because of their lower heat radiation, higher energy efficiency, and longer lifespan compared with fluorescent lamps ( Ma et

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Kui Lin, Zhi Huang, and Yong Xu

contained 20% green light, lettuce showed distinct growth responses to the same irradiance level. Lettuce plants treated with the green-containing LED light showed higher biomass production than those illuminated with red and blue only or the fluorescent

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Yali Liu, Fuling Hao, Rui Meng, and Weirong Xu

The enzyme ACC oxidase (ACO), encoded by a small multigene family in many plants, catalyzes the terminal step in the ethylene biosynthesis pathway. In this research, based on the total RNA isolated from the flowers of Asia hybrids `Pollyanna' and Oriental hybrids `Sorbonne', we obtained two cDNA fragments of ACO genes (Genbank accession DQ062133 and DQ062134) by RT-PCR technique. The two cDNA fragments were reversely inserted into plant expression vector pWR306 respectively, and constructed two antisense ACO gene expression binary vectors harboring hygromycin phosphotransferase (hptII), glucuronidase (uid A), and a green fluorescent protein (GFP) gene in the T-DNA region. We have developed a system to produce transgenic plants in LiLium via Agrobacterium tumefaciens-mediated transformation of calli. Transformants were subjected to GFP expression analysis, PCR assay, and Southern hybridization to confirm gene integration.

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Raquel L. Boscariol, Mariza Monteiro, Elizabete K. Takahashi, Sabrina M. Chabregas, Maria Lucia C. Vieira, Luiz G.E. Vieira, Luiz F.P. Pereira, Francisco de A.A. Mourão Filho, Suane C. Cardoso, Rock S.C. Christiano, Armando Bergamin Filho, Janaynna M. Barbosa, Fernando A. Azevedo, and Beatriz M.J. Mendes

Citrus canker, caused by Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al., is one of the main problems affecting citrus production. In order to obtain resistance to phytopathogenic bacteria, insect genes, coding for antimicrobial proteins, have been used in plant genetic transformation. In this study, transgenic Citrus sinensis (L.) Osb. `Hamlin' plants expressing the antimicrobial insect-derived attacin A gene (attA) were obtained by Agrobacterium tumefaciens (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pCattA 2300). The construction included the native signal peptide (SP) responsible for directing the insect protein to the extracellular space where bacteria is supposed to accumulate in vivo. In order to investigate the native SP effectiveness in a plant model system, onion (Allium cepa L.) epidermal cells were transformed, via biobalistics, using plasmids containing the attA gene with or without SP, fused with the green fluorescent protein gene (pattA 1303 and pSPattA 1303). Fluorescence accumulation surrounding the cells was observed only in tissues transformed with the plasmid containing the gene with SP, indicating the protein secretion to the apoplast. Citrus transformation was confirmed by PCR and Southern blot hybridization analysis in 12 regenerated plants. Transcription of attA gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 106 cfu/mL suspension of the pathogen X. axonopodis pv. citri. Compared to control (non-transformed plant), seven transgenic lines showed a significant reduction in susceptibility to citrus canker. The results obtained here indicate the potential use of antibacterial proteins to protect citrus from bacterial diseases.

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Ahmad A. Omar, Wen-Yuan Song, James H. Graham, and Jude W. Grosser

Citrus canker disease caused by the bacterial pathogen Xanthomonas axonopodis pv. citri is becoming a worldwide problem. Xa21 gene is a member of the Xa21 gene family of rice, which provides broad spectrum Xanthomonas resistance in rice. `Hamlin' sweet orange [Citrus sinensis (L.) Osbeck) is one of the leading commercial cultivars in Florida because of its high yield potential and early maturity. `Hamlin' also has a high regeneration capacity from protoplasts and is often used in transformation experiments. Since the citrus canker pathogen is in the same genus, this gene may have potential to function against canker in citrus. The wild-type Xa21 gene contains an intron, and there are some questions whether dicot plants can process genes containing monocot introns (the cDNA is intron-free). Plasmids DNA, encoding the non-destructive selectable marker EGFP (Enhanced Green Fluorescent Protein) gene and the cDNA of the Xa21 gene were transformed or co-transformed into `Hamlin' orange protoplasts using polyethylene glycol. More than 200 transgenic embryoids were recovered. More than 400 transgenic plants were developed from 75 independent transgenic events. PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in the transgenic plants. Some of the plants have the GFP only. Southern analysis is showing integration of the cDNA into different sites ranges from one to five sites. Western analysis is showing the expression of the cDNA of the Xa21 gene in the transgenic citrus plants. This is the first time that a gene from rice has been stably integrated and expressed in citrus plants. Canker challenge assay is in progress.

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Manjul Dutt, Dennis J. Gray, Zhijian T. Li, Sadanand Dhekney, and Marilyn M. Van Aman

A major drawback to the use of embryogenic cultures for transformation of grapevine is that their ability to undergo genetic transformation is cultivar-dependent. Also, depending on cultivar, embryogenic cultures are difficult to impossible to maintain over time, reducing their utility for use in genetic transformation. An alternative to the use of embryogenic cultures for transformation of grapevine is the use of micropropagation cultures, which are easier to initiate from a wide range of grapevine cultivars and can be maintained over time without loss of function. Vitis vinifera `Thompson Seedless' was used as a model for genetic transformation using micropropagation cultures. In vitro cultures were initiated from apical meristems of actively growing vines and maintained in C2D medium containing 4 μM of 6-benzylaminopurine (C2D4B). Shoot tips and nodes were collected from proliferating in vitro cultures for transformation studies. A variety of wounding techniques, including nicking, sonication, and fragmenting of meristematic tissues was employed in order to enable Agrobacterium infection. We used a construct containing a bidirectional 35S promoter complex with a marker gene composed of a bifunctional fusion between an enhanced green fluorescent protein (EGFP) gene and a neomycin phosphotransferase (NPTII) gene in one direction and a hybrid lytic peptide gene in the other. Transgenic shoots growing in C2D4B medium containing 200 mg·L-1 each of carbenicillin and cefotaxime and 20 mg·L-1 of kanamycin were selected based on GFP fluorescence. Transgenic shoots were rooted and transferred to a greenhouse. To date, 18 transgenic lines have been generated. Details on the transformation procedure will be discussed.