Abstract
The ability of NAD3 (subunit 3 of the NADH ubiquinone oxidoreductase) encoded by unedited nad3 (u-nad3) gene, when targeted into mitochondria, to cause ablation of tapetum was investigated. A gene construct was developed by cloning the coxIV presequence from yeast and the u-nad3 from safflower under the tapetum specific TA29 promoter to target U-NAD3 into mitochondria in the tapetal cell layer. Transgenic tobacco plants were realized with this construct using the Agrobacterium mediated transformation method and confirmed for the presence of the transgene. Among these plants, three were completely sterile and four were semi-sterile. The male sterile plants were morphologically similar to fertile plants, but the anthers were shorter and remained regressed below the stigma surface at anthesis. RT-PCR and western blotting confirmed anther specific expression of u-nad3 in these plants. Stable inheritance of the induced male sterility and its co-segregation with the introduced gene cassette was confirmed in the test cross progeny. This study demonstrates the proof-of-concept that u-nad3 could be used as a candidate to induce transgenic male sterility in plants.
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Abbreviations
- CMS:
-
Cytoplasmic male sterility
- GMS:
-
Genetic male sterility
- CGMS:
-
Cytoplasmic genetic male sterility
- TA29:
-
Tapetum-specific promoter from tobacco
- NADH:
-
Reduced form of nicotinamide adenine dinucleotide
- u-nad3 :
-
Unedited nad3
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Acknowledgments
The financial assistance by Indian Council of Agricultural Research (ICAR), New Delhi through a research Grant (Code No. 030558005) and by Department of Biotechnology, New Delhi through a research Grant (BT/PR9589/AGR/02/441/07) to V.D.K. for carrying out this work is acknowledged. Senior Research Fellowship by ICAR to K.N.Y is acknowledged. The help by Dr. Nagamalleswari, Associate Professor, Ruska lab, ANGRAU, Hyderabad, India in microtomy work and Dr. Rajan Sriraman, Indian Immunologicals Ltd, Hyderabad in carrying out protein work is acknowledged. Authors are thankful to the Project Director, Directorate of Oilseeds Research, Hyderabad for providing all the facilities for carrying out this work. Suggestions and help given by Dr. S. R. Bhat, Principal Scientist, NRCPB, IARI, New Delhi, Dr. M. Sujatha, Principal Scientist, Directorate of Oilseeds Research, Hyderabad and Dr. Nizampatnam NR, DOR, Hyderabad during the course of this investigation are acknowledged. The crucial and critical suggestions by the two anonymous reviewers which helped in improving the quality and clarity of the manuscript are acknowledged.
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Anusha Srinivasan and Kalinati Narasimhan Yamini have contributed equally to this work.
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11240_2014_595_MOESM1_ESM.tif
Supplementary Fig. 1 Southern blotting analysis with male sterile TCNN transformants. Genomic DNA was digested with XhoI and probed with DIG labeled PCR product corresponding a 440 bp fragment comprising coxIV-u-nad3 sequence amplified using specific coxIV F and u-nad3 R primers (Table 1) and was labelled using the non-radio labelling kit (Roche Diagnostics, USA) by following the manufacturer’s instructions. The plant numbers are indicated on the top of the lanes. N1 and N2 correspond to control NX and NT plants respectively. S Sterile plant, SS Semi-sterile, C Control plant. (TIFF 164 kb)
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Srinivasan, A., Yamini, K.N., Reddy, S.S. et al. Tapetum specific expression of unedited nad3 gene from safflower and targeting the protein into mitochondria induces male sterility in transgenic tobacco plants. Plant Cell Tiss Organ Cult 120, 387–398 (2015). https://doi.org/10.1007/s11240-014-0595-0
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DOI: https://doi.org/10.1007/s11240-014-0595-0