Agrobacterium-mediated Tnt1 mutagenesis of moss protonemal filaments and generation of stable mutants with impaired gametophyte

  • Boominathan Mohanasundaram
  • Vyankatesh B. Rajmane
  • Sukanya V. Jogdand
  • Amey J. Bhide
  • Anjan K. BanerjeeEmail author
Original Article


The gametophyte of moss exhibits a simple body plan, yet its growth is regulated by complex developmental phenomena similar to angiosperms. Because moss can be easily maintained under laboratory conditions, amenable for gene targeting and the availability of genome sequence, P. patens has become an attractive model system for studying evolutionary traits. Until date, there has been no Agrobacterium-mediated Tnt1 mutagenesis protocol for haploid protonemal filaments of moss. Hence, we attempted to use the intact tobacco Tnt1 retrotransposon as a mutagen for P. patens. Bioinformatic analysis of initiator methionyl-tRNA (Met-tRNAi), a critical host factor for Tnt1 transposition process, suggested that it can be explored as a mutagen for bryophytes. Using protonemal filaments and Agrobacterium-mediated transformation, 75 Tnt1 mutants have been generated and cryopreserved. SSAP analysis and TAIL-PCR revealed that Tnt1 is functional in P. patens and has a high-preference for gene and GC-rich regions. In addition, LTR::GUS lines exhibited a basal but tissue-specific inducible expression pattern. Forward genetic screen resulted in 5 novel phenotypes related to hormonal and gravity response, phyllid, and gamete development. SSAP analysis suggests that the Tnt1 insertion pattern is stable under normal growth conditions and the high-frequency phenotypic deviations are possibly due to the combination of haploid explant (protonema) and the choice of mutagen (Tnt1). We demonstrate that Agrobacterium-mediated Tnt1 insertional mutagenesis could generate stable P. patens mutant populations for future forward genetic studies.


Physcomitrella patens Moss Forward genetic screen Tnt1 retrotransposon LTR Initiator methionyl-tRNA Gametophyte development 



Critical GC value


Initiator methionyl-tRNA


Primer-binding site

P. patens

Physcomitrella patens


Long-terminal repeats


Transfer DNA


Thermal asymmetric interlaced PCR


Transfer RNA



We sincerely thank Prof. Pascal Ratet (CNRS, France) for providing us tobacco Tnt1 retrotransposon (pCAMBIA-1391Xc-Tnt1) construct. BM acknowledges fellowship support from CSIR, New Delhi. All authors thank IISER Pune and DST Govt. of India for core funding support for this investigation. We are thankful to Prof. Meenu Kapoor for her timely help in moss establishment at IISER Pune. Thanks to Mr. Nitish Lahigude for his support in moss maintenance and Ms. Kavya Mohan for her technical help in cryopreservation of moss mutants. We also thank Prof. David Hannapel, Iowa State University for critical reading of our manuscript.

Author contributions

BM and AKB have conceived and designed the experiments. BM, VR, SJ, and AB have carried out all experiments. BM and AKB have written the manuscript.


The present study was supported by a grant (Grant No. EMR/2016/004852) from Department of Science and Technology (DST), Government of India to AKB. Core funding and infrastructure was provided by Indian Institute of Science Education and Research (IISER) Pune, India.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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  1. Ashton NW, Grimsley NH, Cove DJ (1979) Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants. Planta 144:427–435CrossRefGoogle Scholar
  2. Caine RS, Chater CC, Kamisugi Y et al (2016) An ancestral stomatal patterning module revealed in the non-vascular land plant Physcomitrella patens. Development 143:3306–3314CrossRefGoogle Scholar
  3. Casacuberta JM, Grandbastien M-A (1993) Characterisation of LTR sequences involved in the protoplast specific expression of the tobacco Tnt1 retrotransposon. Nucl Acids Res 21:2087–2093CrossRefGoogle Scholar
  4. Chater CC, Caine RS, Tomek M et al (2016) Origin and function of stomata in the moss Physcomitrella patens. Nat Plants 2:16179CrossRefGoogle Scholar
  5. Coruh C, Cho SH, Shahid S et al (2015) Comprehensive annotation of Physcomitrella patens small RNA loci reveals that the heterochromatic short interfering RNA pathway is largely conserved in land plants. Plant Cell 27:2148–2162CrossRefGoogle Scholar
  6. Courtial B, Feuerbach F, Eberhard S et al (2001) Tnt1 transposition events are induced by in vitro transformation of Arabidopsis thaliana, and transposed copies integrate into genes. Mol Genet Genom 265:32–42CrossRefGoogle Scholar
  7. Cove DJ, Quatrano RS (2006) Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response. Plant Cell Environ 29:1379–1387CrossRefGoogle Scholar
  8. Cove DJ, Perroud P-F, Charron AJ et al (2009) Culturing the moss Physcomitrella patens. Cold Spring Harb Protoc 2009:pdb.prot5136. Google Scholar
  9. Cui Y, Barampuram S, Stacey MG et al (2013) Tnt1 retrotransposon mutagenesis: a tool for soybean functional genomics. Plant Physiol 161:36–47. CrossRefGoogle Scholar
  10. d’Erfurth I, Cosson V, Eschstruth A et al (2003) Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula. Plant J 34:95–106CrossRefGoogle Scholar
  11. Doyle JJ (1990) Isolation of plant DNA from fresh tissue. Focus (Madison) 12:13–15Google Scholar
  12. Duangpan S, Zhang W, Wu Y et al (2013) Insertional mutagenesis using Tnt1 retrotransposon in potato. Plant Physiol 163:21–29. CrossRefGoogle Scholar
  13. Feuerbach F, Drouaud J, Lucas H (1997) Retrovirus-like end processing of the tobacco Tnt1 retrotransposon linear intermediates of replication. J Virol 71:4005–4015Google Scholar
  14. Finnegan DJ (2012) Retrotransposons. Curr Biol 22:R432–R437. CrossRefGoogle Scholar
  15. Fujita T, Sakaguchi H, Hiwatashi Y et al (2008) Convergent evolution of shoots in land plants: lack of auxin polar transport in moss shoots. Evol Dev 10:176–186CrossRefGoogle Scholar
  16. Goodstein DM, Shu S, Howson R et al (2012) Phytozome: a comparative platform for green plant genomics. Nucl Acids Res 40:D1178–D1186CrossRefGoogle Scholar
  17. Grandbastien M-A, Spielmann A, Caboche M (1989) Tnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. Nature 337:376–380CrossRefGoogle Scholar
  18. Hayashida A, Takechi K, Sugiyama M et al (2005) Isolation of mutant lines with decreased numbers of chloroplasts per cell from a tagged mutant library of the moss Physcomitrella patens. Plant Biol 7:300–306CrossRefGoogle Scholar
  19. Hori K, Maruyama F, Fujisawa T et al (2014) Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation. Nat Commun 5Google Scholar
  20. Hunter JD (2007) Matplotlib: a 2D graphics environment. Comput Sci Eng 9:90–95CrossRefGoogle Scholar
  21. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901CrossRefGoogle Scholar
  22. Jenkins GI, Cove DJ (1983a) Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of mutant strains. Planta 159:432–438. CrossRefGoogle Scholar
  23. Jenkins GI, Cove DJ (1983b) Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of the wild-type. Planta 158:357–364CrossRefGoogle Scholar
  24. Jenkins GI, Courtice GRM, Cove DJ (1986) Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens. Plant Cell Environ 9:637–644CrossRefGoogle Scholar
  25. Kartha KK, Engelmann F (1994) Cryopreservation and germplasm storage. In: Vasil IK, Thorpe TA (eds) Plant cell and tissue culture. Springer, Dordrecht, pp 195–230Google Scholar
  26. Kim Sang-Ic, Gelvin stanton B (2007) Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions. Plant J 51:779–791. doiCrossRefGoogle Scholar
  27. Kofuji R, Hasebe M (2014) Eight types of stem cells in the life cycle of the moss Physcomitrella patens. Curr Opin Plant Biol 17:13–21CrossRefGoogle Scholar
  28. Lang D, Eisinger J, Reski R, Rensing SA (2005) Representation and high-quality annotation of the Physcomitrella patens transcriptome demonstrates a high proportion of proteins involved in metabolism in mosses. Plant Biol 7:238–250CrossRefGoogle Scholar
  29. Lucas H, Feuerbach F, Kunert K et al (1995) RNA-mediated transposition of the tobacco retrotransposon Tnt1 in Arabidopsis thaliana. EMBO J 14:2364CrossRefGoogle Scholar
  30. Mhiri C, Morel J-B, Vernhettes S et al (1997) The promoter of the tobacco Tnt1 retrotransposon is induced by wounding and by abiotic stress. Plant Mol Biol 33:257–266CrossRefGoogle Scholar
  31. Nishiyama T, Hiwatashi Y, Sakakibara K et al (2000) Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res 7:9–17CrossRefGoogle Scholar
  32. Pavesi A, Conterio F, Bolchi A et al (1994) Identification of new eukaryotic tRNA genes in genomic DNA databases by a multistep weight matrix anaylsis of transcriptional control regions. Nucl Acids Res 22:1247–1256CrossRefGoogle Scholar
  33. Pouteau S, Huttner E, Grandbastien MA, Caboche M (1991) Specific expression of the tobacco Tnt1 retrotransposon in protoplasts. EMBO J 10:1911CrossRefGoogle Scholar
  34. Pouteau S, Grandbastien M-A, Boccara M (1994) Microbial elicitors of plant defence responses activate transcription of a retrotransposon. plant J 5:535–542CrossRefGoogle Scholar
  35. Prigge MJ, Bezanilla M (2010) Evolutionary crossroads in developmental biology: Physcomitrella patens. Development 137:3535–3543CrossRefGoogle Scholar
  36. Sakakibara K, Nishiyama T, Deguchi H, Hasebe M (2008) Class 1 KNOX genes are not involved in shoot development in the moss Physcomitrella patens but do function in sporophyte development. Evol Dev 10:555–566CrossRefGoogle Scholar
  37. Sakakibara K, Ando S, Yip HK et al (2013) KNOX2 genes regulate the haploid-to-diploid morphological transition in land plants. Science 339:1067–1070CrossRefGoogle Scholar
  38. Sambrook J, Fritsch EF, Maniatis T et al (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  39. Schaefer DG, Zrÿd J-P (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11:1195–1206. CrossRefGoogle Scholar
  40. Schulte J, Reski R (2004) High throughput cryopreservation of 140 000 Physcomitrella patens mutants. Plant Biol 6:119–127CrossRefGoogle Scholar
  41. Stevenson SR, Kamisugi Y, Trinh CH et al (2016) Genetic analysis of Physcomitrella patens identifies ABSCISIC ACID NON-RESPONSIVE (ANR), a regulator of ABA responses unique to basal land plants and required for desiccation tolerance. Plant Cell 28:1310–1327Google Scholar
  42. Syed NH, Flavell AJ (2006) Sequence-specific amplification polymorphisms (SSAPs): a multi-locus approach for analyzing transposon insertions. Nat Protoc 1:2746CrossRefGoogle Scholar
  43. Tam SM, Mhiri C, Vogelaar A et al (2005) Comparative analyses of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR. Theor Appl Genet 110:819–831CrossRefGoogle Scholar
  44. Tanahashi T, Sumikawa N, Kato M, Hasebe M (2005) Diversification of gene function: homologs of the floral regulator FLO/LFY control the first zygotic cell division in the moss Physcomitrella patens. Development 132:1727–1736CrossRefGoogle Scholar
  45. Vernhettes S, Grandbastien M-A, Casacuberta JM (1997) In vivo characterization of transcriptional regulatory sequences involved in the defence-associated expression of the tobacco retrotransposon Tnt1. Plant Mol Biol 35:673–679CrossRefGoogle Scholar
  46. Vives C, Charlot F, Mhiri C et al (2016) Highly efficient gene tagging in the bryophyte Physcomitrella patens using the tobacco (Nicotiana tabacum) Tnt1 retrotransposon. New Phytol 212:759–769CrossRefGoogle Scholar
  47. Walt S van der, Colbert SC, Varoquaux G (2011) The NumPy array: a structure for efficient numerical computation. Comput Sci Eng 13:22–30CrossRefGoogle Scholar
  48. Waugh R, McLean K, Flavell AJ et al (1997) Genetic distribution of Bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol Gen Genet MGG 253:687–694CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Indian Institute of Science Education and Research (IISER, Pune)PuneIndia

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