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Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato

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Abstract

Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) are essential component of every cell because of their involvement in the regulation of cell division, growth and development. The aim of this study is to enhance the levels of Put during fruit development and see its implications in ripening and quality of tomato fruits. Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) were developed. Transgenic fruits exhibited enhanced levels of Put, Spd and Spm, with a concomitant reduction in ethylene levels, rate of respiration and physiological loss of water. Consequently such fruits displayed significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. The activation of Put biosynthetic pathway at the onset of ripening in transgenic fruits is also consistent with the improvement of qualitative traits such as total soluble solids, titratable acids and total sugars. Such changes were associated with alteration in expression pattern of ripening specific genes. Transgenic fruits were also fortified with important nutraceuticals like lycopene, ascorbate and antioxidants. Therefore, these transgenic tomatoes would be useful for the improvement of tomato cultivars through breeding approaches.

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Acknowledgments

We express our gratitude to Prof. Tony Pegg (Hershey, USA) for providing the m-ODC cDNA and Dr. V. S. Reddy (International Centre for Genetic Engineering and Biotechnology, New Delhi) for providing the 2A11 tomato promoter. This work was generously supported by Grants from the Department of Biotechnology (Govt. of India), New Delhi (Grant Nos. BT/PR/2990/Agr/16/232/2002 and BT/PR8657/PBD/16/738/2007). We also thank the University Grants Commission, New Delhi for Special Assistance Programme and Department of Science and Technology, New Delhi for FIST and DU-DST PURSE programmes. Research fellowships to Roopali Pandey and Aarti Gupta by the Council of Scientific and Industrial Research are acknowledged.

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The authors declare that they have no conflict of interests.

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Authors and Affiliations

  1. Plant Polyamine, Transgenic and RNAi Research Laboratory, Department of Genetics, University of Delhi South Campus, New Delhi, 110021, India

    Roopali Pandey, Aarti Gupta & Manchikatla Venkat Rajam

  2. Division of Post-Harvest Technology, Indian Agricultural Research Institute, New Delhi, 110012, India

    Anuj Chowdhary & Ram Krishna Pal

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  1. Roopali Pandey
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  2. Aarti Gupta
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  3. Anuj Chowdhary
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  4. Ram Krishna Pal
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  5. Manchikatla Venkat Rajam
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Correspondence to Manchikatla Venkat Rajam.

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Roopali Pandey and Aarti Gupta have contributed equally to the article.

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Supplementary Fig. 1 T-DNA map of m-ODC gene construct. (JPEG 13 kb)

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Supplementary Fig. 2 Molecular characterization and segregation analysis of tomato transformants. (a) PCR analysis with primers specific to NPT-II gene in primary transformants: L - 1 kb ladder; PC - Plasmid DNA; UT - DNA from untransformed control; LeODC 22-72 - DNA from different transgenic tomato lines. (b) Southern blot analysis of T0 generation of LeODC transgenic plants, revealing transgene integration and copy number using m-ODC gene radio-labeled probe. UT - DNA from untransformed control; LeODC 11-60- DNA from different transgenic lines digested with Xba I enzyme. (c) The leaves of T1 transgenic line LeODC22 showing segregation of the m-ODC transgene on kanamycin (50 mg/l) amended MS basal medium, and this was based on the bleaching of leaves. The untransformed leaves (UT) showed complete bleaching on antibiotic medium. (d1 and d2). PCR analysis with primers specific to m-ODC gene showing segregation pattern of transgene in T2 and T3 generations respectively. L - 1 kb ladder; PC - Plasmid DNA, UT-DNA from untransformed control; B - blank, 1-12 - DNA from different progenies of LeODC27 line. (e) Southern blot analysis of T3 generation of LeODC transgenic plants revealing homozygous status of integrated transgene, using m-ODC gene radio-labeled probe. LeODC22-LeODC32 - DNA from T3 progenies of different transgenic lines digested with XbaI enzyme. (JPEG 90 kb)

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Supplementary Fig. 3 A lineage chart illustrating analysis of segregation patterns across different generations. Leaves from 100 T1 seedlings (named as e.g., LeODC22-1, LeODC22-2……. LeODC22-100) of each positive T0 plant (e.g. LeODC22) were screened by kanamycin resistant assay and 40 positive segregants were further confirmed by m-ODC gene specific PCR. All the homozygous lines followed 3:1 transgene segregation pattern in T1 generation. Ten positive segregants were taken to raise T2 generation. Hundred T2 seedlings (further named as e.g., LeODC22-1.1, LeODC22-1.2……. LeODC22-1.100.) from each of 10 such parents were further screened by kanamycin resistance assay and confirmed by PCR. (JPEG 72 kb)

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Supplementary Fig. 4 Pollen viability UT and transgenic tomato lines. Pollen viability was scored based on pollen germination after 2 h incubation in germination media in dark. Data is the representation of T3 generation showing means of 500 pollen counts. Each experiment was repeated thrice in four generations (T0-T3) with reproducible results. (JPEG 12 kb)

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Supplementary Fig. 5 Semi-quantitative RT-PCR analysis showing expression patterns of fruit specific genes. (a) Expression pattern of PA biosynthesis genes; SlODC, SlADC, SlSAMDC1 and SlSPDSYN, (b) Expression pattern of ethylene biosynthesis genes; SlACS2, SlACS4 and SlACO1, (c) Expression pattern of cell wall hydrolyzing genes; SlEXP1, SlTBG4, SlPG2a and SlXTH (d) Expression pattern of lycopene biosynthesis genes; SlPSY1, SlDXS1 and SlLES; in transgenic (T3 generation) and UT fruits at different stages (MG, BR, PR and RR) of ripening. SlACTIN gene expression was maintained as internal control. Expression analysis was done thrice using 50 ng of total RNA, with ‘n’ no. of cycles utilizing three biological replicates. (JPEG 69 kb)

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Pandey, R., Gupta, A., Chowdhary, A. et al. Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato. Plant Mol Biol 87, 249–260 (2015). https://doi.org/10.1007/s11103-014-0273-y

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