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, 21 (3), 401-10

Plastid Transformation in the Monocotyledonous Cereal Crop, Rice (Oryza Sativa) and Transmission of Transgenes to Their Progeny

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Plastid Transformation in the Monocotyledonous Cereal Crop, Rice (Oryza Sativa) and Transmission of Transgenes to Their Progeny

Sa Mi Lee et al. Mol Cells.

Abstract

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.

Figures

Fig. 1
Fig. 1
Rice transplastome resulting from the homologous recombination between the rice plastid transformation vector, pLD-RCtV-sGFP, and rice plastid DNA. The aadA and sgfp genes are driven by the rRNA operon promoter (Prrn). TpsbA indicates the 3′ UTR of the psbA gene. The filled triangles indicate the primer sites used for PCR analyses. The probe used for Southern analysis is marked with a thick line.
Fig. 2
Fig. 2
Determination of the optimal streptomycin concentration for inhibition of callus growth and shoot induction. The growth response of the total calli (2–3 weeks after germination) on selective regeneration medium containing (A) 0 (left), 100 (middle) and 200 (right) mg/L of streptomycin, after 2 months of culture in the light condition, and (B) 0 (left), 500 (middle) and 1,000 (right) mg/L of streptomycin, after 1 month of culture in the dark condition.
Fig. 3
Fig. 3
Recovery of transplastomic rice plants. (A) shoots induced from the bombarded calli after 2 months’ incubation on regeneration medium containing 200 mg/L of streptomycin, (B) streptomycin-resistant shoots regenerated into a plantlet, approximately 1 month after transferring to rooting medium containing 500 mg/L of streptomycin and (C) phenotype of the mature transplastomic plants in the greenhouse, indicating streptomycin resistance at different concentrations.
Fig. 4
Fig. 4
Characterization of T0 transplastomic plants. (A) PCR analysis indicates the presence of the 590 bp sgfp gene fragment in all six of the streptomycin-resistant plants derived from sGFP-1 and sGFP-2; streptomycin concentrations used in these selections are shown in parentheses, (B) Southern analysis using the aadA probe detects the 3.3 kb BamHI fragment in both sGFP-1 and sGFP-2, (C) PCR amplification of 1.9 and 1.3 kb left and right border DNA fragments, respectively and (D) the detection of sGFP accumulation via Western analysis.
Fig. 5
Fig. 5
T1 progeny analyses. (A) Plastid-localized sGFP expression (marked with arrows) is observed from transplastomic line (top), whereas only a background level of autofluorescence is detectable from non-transformed plant (bottom) and (B) PCR analysis showing the presence of the 538 and 221 bp fragments, representing the transplastome and untransplastome, respectively.

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