The miR408 expression in scutellum derived somatic embryos of Oryza sativa L. ssp. indica varieties: media and regenerating embryos

  • Manish Solanki
  • Anshika Sinha
  • Lata I. ShuklaEmail author
Original Article


The conserved miRNAs are key players for post-transcriptional control of gene expression for plant growth and development. The overexpression of miR408 is reported to show improvement in biomass and photosynthesis in Oryza sativa. Also, microarray data show differential expression in somatic and regenerating embryos of japonica rice. The position weight matrix analyses of miR408 (72 sequences) family in plant kingdom was performed using DAMBE and targets were obtained by psRNATarget. The qRT-PCR experiments were performed using stem-loop primers to evaluate the role of miR408-3p and targets in seven different indica rice varieties, the formation of regenerating pluripotent somatic embryos and assessment of suitable conditions for generation of somatic embryos. Different callus induction media and regeneration media, healthy calli, brown calli and necrotic calli were also used to assess the expression of miR408-3p and its targets. The conserved sequence of miR408-3p was observed and 68 targets were identified using psRNATarget in Oryza sativa. The qRT-PCR was performed on target Uclacyanin which shows mismatch at position 14 and 15, and RNA polymerase II at position 14 and 17. The miR408-3p expression shows recalcitrant > moderately recalcitrant > non-recalcitrant in indica rice variety. Significant differences in the expression of miR408-3p for the developing somatic embryos at 10-day, 15-day, 20-day, 25-day and 30-day old somatic embryos were observed. Evidences for miR408-3p expression and identification of new target RNA Polymerase II is shown. The lower expression of miR408-3p shows parallelism with the generation of healthy somatic embryos with applications for rapid screening of pluripotency in Oryza sativa.


miR408 Uclacyanin RNA Pol II Somatic embryo Indica rice 



MS, AS and LIS grateful to DBT, GOI for research grant BT/PR11797/AGR/36/608/2009 and DST-FIST SR/FST/LSI-366/2008 Dt. 18.02.2009. MS thank CSIR-UGC India for providing SRF, AS thank UGC-BSR India for providing SRF. Mohammed Javed is gratefully acknowledged for helping in position weight matrix analysis.

Author contributions

MS is the first author has performed the somatic embryo generation, qRT-PCR experiments, data analysis and has written the manuscript. AS is the second author has performed the regeneration experiments of somatic embryo and position weight matrix analysis. LIS has supervised the experiments and has corrected the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (DOCX 1209 kb)
11240_2019_1602_MOESM2_ESM.docx (64 kb)
Supplementary material 2 (DOCX 63 kb)


  1. Abdel-Ghany SE, Pilon M (2008) MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis. J Biol Chem 283(23):15932–15945. CrossRefGoogle Scholar
  2. Anshika & Shukla LI (2015) Identification of essential constituents for development of embryogenic non-recalcitrant calli from recalcitrant indica rice variety CR1009 and ASD16. GSTF e-Journal on ICT:44–49
  3. Axtell MJ, Bowman JL (2008) Evolution of plant microRNAs and their targets. Trends Plant Sci 13(7):343–349. CrossRefGoogle Scholar
  4. Brancati G, Großhans H (2018) An interplay of miRNA abundance and target site architecture determines miRNA activity and specificity. Nucleic Acids Res 46(7):3259–3269. CrossRefGoogle Scholar
  5. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179. CrossRefGoogle Scholar
  6. Chen CJ, liu Q, Zhang YC, Qu LH, Chen YQ, Gautheret D (2011) Genome-wide discovery and analysis of microRNAs and other small RNAs from rice embryogenic callus. RNA Biol 8(3):538–547CrossRefGoogle Scholar
  7. Chu CC (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci Sin 18:659–668Google Scholar
  8. Chuanjun X, Zhiwei R, Ling L, Biyu Z, Junmei H, Wen H, Ou H (2015) The effects of polyphenol oxidase and cycloheximide on the early stage of browning in phalaenopsis explants. Hortic Plant J 1(3):172–180. Google Scholar
  9. Cuperus JT, Fahlgren N, Carrington JC (2011) Evolution and functional diversification of MIRNA genes. Plant Cell 23(2):431–442. CrossRefGoogle Scholar
  10. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151–158CrossRefGoogle Scholar
  11. Javed M, Solanki M, Sinha A, Shukla LI (2017) Position based nucleotide analysis of mir168 family in higher plants and its targets in mammalian transcripts. MicroRNA 6(2):136–142. CrossRefGoogle Scholar
  12. Javed M, Sinha A, Israni Shukla L (2018) Evaluation of mature miR398 family, expression analysis and the post transcriptional regulation evidence in gamma-irradiated and nitrogen stressed Medicago sativa seedlings. Int J Rad Biol 1:1–36. CrossRefGoogle Scholar
  13. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53. CrossRefGoogle Scholar
  14. Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Research 42(Database issue):D68–73. CrossRefGoogle Scholar
  15. Lee K, Seo PJ (2018) Dynamic epigenetic changes during plant regeneration. Trends Plant Sci 23(3):235–247. CrossRefGoogle Scholar
  16. Liang G, He H, Yu D (2012) Identification of nitrogen starvation-responsive microRNAs in Arabidopsis thaliana. PLoS ONE 7(11):e48951. CrossRefGoogle Scholar
  17. Liang G, Ai Q, Yu D (2015) Uncovering miRNAs involved in crosstalk between nutrient deficiencies in Arabidopsis. Sci Rep 5:11813. CrossRefGoogle Scholar
  18. Lin YJ, Zhang Q (2005) Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep 23(8):540–547. CrossRefGoogle Scholar
  19. Liu B, Li P, Li X, Liu C, Cao S, Chu C, Cao X (2005) Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice. Plant Physiol 139(1):296–305. CrossRefGoogle Scholar
  20. Ma C, Burd S, Lers A (2015) miR408 is involved in abiotic stress responses in Arabidopsis. Plant J 84(1):169–187. CrossRefGoogle Scholar
  21. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  22. Mutum RD, Balyan SC, Kansal S, Agarwal P, Kumar S, Kumar M, Raghuvanshi S (2013) Evolution of variety-specific regulatory schema for expression of osa-miR408 in indica rice varieties under drought stress. FEBS J 280(7):1717–1730. CrossRefGoogle Scholar
  23. Pan J, Huang D, Guo Z, Kuang Z, Zhang H, Xie X, Ma Z, Gao S, Lerdau MT, Chu C, Li L (2018) Overexpression of microRNA408 enhances photosynthesis, growth, and seed yield in diverse plants. J Integr Plant Biol 60(4):323–340. CrossRefGoogle Scholar
  24. Paul S, Datta SK, Datta K (2015) miRNA regulation of nutrient homeostasis in plants. Front Plant Sci 6:232. CrossRefGoogle Scholar
  25. Sah S, Kaur A, Kaur G, Cheema G (2014) Genetic transformation of rice: problems. Progr Prospects J Rice Res 3:132. Google Scholar
  26. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108CrossRefGoogle Scholar
  27. Shoeb F, Yadav JS, Bajaj S, Rajam MV (2001) Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice. Plant Sci 160(6):1229–1235CrossRefGoogle Scholar
  28. Sinha A, Solanki M, Shukla LI (2018) Evidences for differential expression of miR167d-5p, target, positional nucleotide preference, and its role in somatic and different stages of regenerating calli of Oryza sativa. Plant Cell Tissue Organ Cult 136(3):537–548. CrossRefGoogle Scholar
  29. Song Z, Zhang L, Wang Y, Li H, Li S, Zhao H, Zhang H (2018) Constitutive expression of miR408 improves biomass and seed yield in Arabidopsis. Front Plant Sci 8:2114. CrossRefGoogle Scholar
  30. Sunkar R, Zhu J-K (2004) Novel and stress-regulated micrornas and other small rnas from Arabidopsis. Plant Cell 16(8):2001–2019. CrossRefGoogle Scholar
  31. Wang Y, Zhang C, Hao Q, Sha A, Zhou R, Zhou X, Yuan L (2013) Elucidation of miRNAs-mediated responses to low nitrogen stress by deep sequencing of two soybean genotypes. PLoS ONE 8(7):e67423. CrossRefGoogle Scholar
  32. Xia X (2013) DAMBE5: a comprehensive software package for data analysis in molecular biology and evolution. Mol Biol Evol 30(7):1720–1728. CrossRefGoogle Scholar
  33. Xu Y, Zhang L, Wu G (2018) Epigenetic regulation of juvenile-to-adult transition in plants. Front Plant Sci 9:1048–1048. CrossRefGoogle Scholar
  34. Xue LJ, Zhang JJ, Xue HW (2009) Characterization and expression profiles of miRNAs in rice seeds. Nucleic Acids Res 37(3):916–930. CrossRefGoogle Scholar
  35. Yamasaki H, Abdel-Ghany SE, Cohu CM, Kobayashi Y, Shikanai T, Pilon M (2007) Regulation of copper homeostasis by micro-RNA in Arabidopsis. J Biol Chem 282(22):16369–16378. CrossRefGoogle Scholar
  36. Zhang YC, Yu Y, Wang CY, Li ZY, Liu Q, Xu J, Liao JY, Wang XJ, Qu LH, Chen F, Xin P, Yan C, Chu J, Li HQ & Chen YQ (2013) Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nat Biotechnol 31(9):848–852. CrossRefGoogle Scholar
  37. Zhang JP, Yu Y, Feng YZ, Zhou YF, Zhang F, Yang YW, Lei MQ, Zhang YC, Chen YQ (2017) MiR408 regulates grain yield and photosynthesis via a phytocyanin protein. Plant Physiol 175(3):1175–1185. CrossRefGoogle Scholar
  38. Zhao M, Tai H, Sun S, Zhang F, Xu Y, Li WX (2012) Cloning and characterization of maize miRNAs involved in responses to nitrogen deficiency. PLoS ONE 7(1):e29669. CrossRefGoogle Scholar
  39. Zhao XY, Hong P, Wu JY, Chen XB, Ye XG, Pan YY, Wang J, Zhang XS (2016) The tae-miR408-mediated control of TaTOC1 genes transcription is required for the regulation of heading time in wheat. Plant Physiol 170(3):1578–1594. Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Manish Solanki
    • 1
  • Anshika Sinha
    • 1
  • Lata I. Shukla
    • 1
    Email author
  1. 1.Department of Biotechnology, School of Life SciencesPondicherry UniversityKalapetIndia

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