DIANTHIN, a negative selection marker in tobacco, is non-toxic in transgenic rice and confers sheath blight resistance
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The DIANTHIN gene encoding a ribosome-inactivating protein (RIP) from Dianthus caryophyllus L. was tested for negative selection in tobacco and rice. Tobacco leaf discs and scutellum-derived callus of rice were transformed with Agrobacterium tumefaciens strain LBA4404 (pSB1, pJAS1). pJAS1 harbors the DIANTHIN gene under the control of the CaMV 35S promoter. Tobacco transformation efficiency, in comparison to pCAMBIA1301, was reduced by 87 % in pJAS1-transformed leaf discs. The DIANTHIN gene proved to be completely toxic to tobacco as all the recovered hygromycin-resistant transgenic plants harbored truncated T-DNAs with deletions of the DIANTHIN gene. Transformation of the DIANTHIN gene under a Mungbean yellow mosaic virus (MYMV)-inducible promoter did not cause any toxicity in tobacco as reflected by the recovery of transgenic tobacco plants with the complete DIANTHIN gene. Transformation efficiency of pJAS1 did not decline in rice. Interestingly, all transgenic rice plants harbored the complete DIANTHIN gene and expressed the gene. The T1 transgenic lines showed reduction of sheath blight symptom in the range of 29 to 42 %. The difference in the sensitivity to DIANTHIN between tobacco and rice provides a new direction to study the mechanisms underlying RIP toxicity in plants.
Additional key wordsNicotiana tabacum Oryza sativa Rhizoctonia solani ribosome-inactivating protein truncated T-DNA
β-glucuronidase gene with an intron
hygromycin phosphotransferase gene
Murashige and Skoog
negative selection marker
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We gratefully acknowledge Dr. John Stanley, UK, for providing the DIANTHIN gene. We thank Dr. Toshihiko Komari, Japan Tobacco Inc., Japan, for providing pSB1 and Dr. M. Samiappan, Tamil Nadu Agricultural University, Coimbatore, India for providing the Rhizoctonia solani strain RS7. We thank Dr. K. Dharmalingam for permitting us to use his instrumentation facilities. R. Rajeswaran is acknowledged for constructing pRAJ18 and for performing tobacco transformation with pRAJ18. This research was supported by funds from the Department of Biotechnology (DBT) and University Grants Commission (UGC), Government of India. JMS is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for Fellowship.
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