The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T2 generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.

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Soybean [Glycine max (L.) Merr.] is an important crop for vegetable oil production, and is a major protein source worldwide. Because of its importance as a crop, genetic transformation has been used extensively to improve its valuable traits. Soybean mosaic virus (SMV) is one of the most wellknown viral diseases affecting soybean. Transgenic soybean plants with improved resistance to SMV were produced by introducing HC-Pro coding sequences within RNA interference (RNAi) inducing hairpin construct via Agrobacteriummediated transformation. During an experiment to confirm the response of transgenic plants (T 2 ) to SMV infection, no T 2 plants from lines #2 (31/31), #5 (35/35) or #6 (37/37) exhibited any SMV symptoms, indicating strong viral resistance (R), whereas NT (non-transgenic wild type) plants and those from lines #1, #3 and #4 exhibited mild mosaic (mM) or mosaic (M) symptoms. The northern blot analysis showed that three resistant lines (#2, #5 and #6) did not show the detection of viral RNA accumulation while NT, EV (transformed with empty vector carrying only Bar) and lines #1, #3 and #4 plants were detected. T 3 seeds from SMV-inoculated T 2 plants were harvested and checked for changes in seed morphology due to viral infection. T 3 seeds of lines #2, #5 and #6 were clear and seed coat mottling was not present, which is indicative of SMV resistance. RT-PCR and quantitative real-time PCR showed that T 3 seeds from the SMV-resistant lines #2, #5 and #6 did not exhibit any detection of viral RNA accumulation (HC-Pro, CP and CI), while the viral RNA accumulation was detected in SMV-susceptible lines #1, #3 and #4 plants. During the greenhouse test for viral resistance and yield components, T 3 plants from the SMV-inoculated transgenic lines #2, #5 and #6 showed viral resistance (R) and exhibited a more favorable average plant height, number of nodes per plant, number of branches per plant, number of pods per plant and total seed weight with statistical significance during strong artificial SMV infection than did other plant lines. In particular, the SMVresistant line #2 exhibited superior average plant height, pod number and total seed weight with highly significance. According to our results, RNAi induced by the hairpin construct of the SMV HC-Pro sequence effectively confers much stronger viral resistance than did the methods used during previous trials, and has the potential to increase yields significantly. Because of its efficiency, the induction of RNAi-mediated resistance will likely be used more frequently as part of the genetic engineering of plants for crop improvement.

Plants have adapted to environmental challenges by expressing many plant genes in response to the stresses. Among those genes, CCCH zinc finger proteins are involved in abiotic and biotic stresses. Transgenic soybean plants overexpressing AtSZF2 were produced to investigate that its ectopic overexpression enhanced salt stress tolerance by Agrobacterium-mediated transformation using half-seed explants. Sixteen transgenic lines were chosen to analyze for T-DNA insertion and transcription levels, and most of them were confirmed as positive. In further analysis with Southern blot, stable transformation event and copy number were confirmed. Following high salinity stress on the detached leaf and whole plant of two transgenic lines (#4 and #6) revealed that the ectopic expression of AtSZF2 was correlated with stress tolerance in phenotype, ion leakage and chlorophyll content with statistical significance. In another test with 20% PEG treatment, similar tolerance of transgenic plants was observed with lower ion leakage and higher chlorophyll content, indicating that the damage of cell membrane was prevented in transgenic plants. Finally, expression of various abiotic stress-responding genes was detected by reverse transcriptase and quantitative real-time PCR analysis with the transgenic plants. It could be proposed that introduction of AtSZF2 resulted in the modulation of ABA/stress responsive gene expression in transgenic soybean plants and make them tolerant against salt stress. Considering soybean as a salt-sensitive crop and importance of salt stress tolerance in specific farming region, the introduction of AtSZF2 may provide an approach for crop improvement in soybean breeding.

Plants have adapted to environmental challenges by expressing many plant genes in response to the stresses. Among those genes, CCCH zinc finger proteins are involved in abiotic and biotic stresses. Transgenic soybean plants overexpressing AtSZF2 were produced to investigate that its ectopic overexpression enhanced salt stress tolerance by Agrobacterium-mediated transformation using half-seed explants. Sixteen transgenic lines were chosen to analyze for T-DNA insertion and transcription levels, and most of them were confirmed as positive. In further analysis with Southern blot, stable transformation event and copy number were confirmed. Following high salinity stress on the detached leaf and whole plant of two transgenic lines (#4 and #6) revealed that the ectopic expression of AtSZF2 was correlated with stress tolerance in phenotype, ion leakage and chlorophyll content with statistical significance. In another test with 20% PEG treatment, similar tolerance of transgenic plants was observed with lower ion leakage and higher chlorophyll content, indicating that the damage of cell membrane was prevented in transgenic plants. Finally, expression of various abiotic stress-responding genes was detected by reverse transcriptase and quantitative real-time PCR analysis with the transgenic plants. It could be proposed that introduction of AtSZF2 resulted in the modulation of ABA/stress responsive gene expression in transgenic soybean plants and make them tolerant against salt stress. Considering soybean as a salt-sensitive crop and importance of salt stress tolerance in specific farming region, the introduction of AtSZF2 may provide an approach for crop improvement in soybean breeding.

A full-length cDNA of the OgPAE1 gene encoding the alpha5 subunit of the 20S proteasome was isolated from wild rice (Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism.

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