Journal of Plant Research

, Volume 131, Issue 2, pp 349–358 | Cite as

Agrobacterium-mediated floral dip transformation of the model polyploid species Arabidopsis kamchatica

  • Chow-Lih Yew
  • Hiroyuki Kakui
  • Kentaro K. ShimizuEmail author
Technical Note


Polyploidization has played an important role in the speciation and diversification of plant species. However, genetic analyses of polyploids are challenging because the vast majority of the model species are diploids. The allotetraploid Arabidopsis kamchatica, which originated through the hybridization of the diploid Arabidopsis halleri and Arabidopsis lyrata, is an emerging model system for studying various aspects of polyploidy. However, a transgenic method that allows the insertion of a gene of interest into A. kamchatica is still lacking. In this study, we investigated the early development of pistils in A. kamchatica and confirmed the formation of open pistils in young flower buds (stages 8–9), which is important for allowing Agrobacterium to access female reproductive tissues. We established a simple Agrobacterium-mediated floral dip transformation method to transform a gene of interest into A. kamchatica by dipping A. kamchatica inflorescences bearing many young flower buds into a 5% sucrose solution containing 0.05% Silwet L-77 and Agrobacterium harboring the gene of interest. We showed that a screenable marker comprising fluorescence-accumulating seed technology with green fluorescent protein was useful for screening the transgenic seeds of two accessions of A. kamchatica subsp. kamchatica and an accession of A. kamchatica subsp. kawasakiana.


Agrobacterium-mediated floral dip Allopolyploid Arabidopsis kamchatica Tetraploid Transformation Transgenic plant 



We would like to thank Misako Yamazaki, Reiko Akiyama, Yuanyuan Huang, and Rie Shimizu-Inatsugi for technical support and discussion. This research was supported by the Swiss National Science Foundation,, JST CREST Grant Number JPMJCR16O3, Japan, and a KAKENHI Grant (nos. 16H06469, 16H06464, 16K21727).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

10265_2017_982_MOESM1_ESM.pdf (6.8 mb)
Supplementary material 1 (PDF 7009 KB)


  1. Abell BM, Holbrook LA, Abenes M, Murphy DJ, Hills MJ, Moloney MM (1997) Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting. Plant Cell 9:1481–1493. doi: 10.1105/tpc.9.8.1481 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Akama S, Shimizu-Inatsugi R, Shimizu KK, Sese J (2014) Genome-wide quantification of homeolog expression ratio revealed nonstochastic gene regulation in synthetic allopolyploid Arabidopsis. Nucleic Acids Res 42:e46. doi: 10.1093/nar/gkt1376 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Armstrong JJ, Takebayashi N, Sformo T, Wolf DE (2015) Cold tolerance in Arabidopsis kamchatica. Am J Bot 102:439–448. doi: 10.3732/ajb.1400373 CrossRefPubMedGoogle Scholar
  4. Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–266. doi: 10.1385/0-89603-391-0:259 PubMedGoogle Scholar
  5. Bressan RA, Zhang CQ, Zhang H, Hasegawa PM, Bohnert HJ, Zhu JK (2001) Learning from the Arabidopsis experience. The next gene search paradigm. Plant Physiol 127:1354–1360. doi: 10.1104/Pp.010752 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Briskine RV, Paape T, Shimizu-Inatsugi R, Nishiyama T, Akama S, Sese J, Shimizu KK (2016) Genome assembly and annotation of Arabidopsis halleri, a model for heavy metal hyperaccumulation and evolutionary ecology. Mol Ecol Resour. doi: 10.1111/1755-0998.12604 PubMedGoogle Scholar
  7. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743. doi: 10.1046/J.1365-313x.1998.00343.X CrossRefPubMedGoogle Scholar
  8. D’Halluin K, De Block M, Denecke J, Janssens J, Leemans J, Reynaerts A, Botterman J (1992) The bar gene as selectable and screenable marker in plant engineering. Methods Enzymol 216:415–426. doi: 10.1016/0076-6879(92)16038-L CrossRefPubMedGoogle Scholar
  9. Desfeux C, Clough SJ, Bent AF (2000) Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol 123:895–904. doi: 10.1104/pp.123.3.895 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fobis-Loisy I, Chambrier P, Gaude T (2007) Genetic transformation of Arabidopsis lyrata: specific expression of the green fluorescent protein (GFP) in pistil tissues. Plant Cell Rep 26:745–753. doi: 10.1007/s00299-006-0282-7 CrossRefPubMedGoogle Scholar
  11. Fujimoto R, Kinoshita Y, Kawabe A, Kinoshita T, Takashima K, Nordborg M, Nasrallah ME, Shimizu KK, Kudoh H, Kakutani T (2008) Evolution and control of imprinted FWA genes in the genus Arabidopsis. PLoS Genet 4:21000048. doi: 10.1371/journal.pgen.1000048 CrossRefGoogle Scholar
  12. Hanikenne M, Talke IN, Haydon MJ, Lanz C, Nolte A, Motte P, Kroymann J, Weigel D, Kramer U (2008) Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4. Nature 453:391–395. doi: 10.1038/nature06877 CrossRefPubMedGoogle Scholar
  13. Hay AS, Pieper B, Cooke E, Mandakova T, Cartolano M, Tattersall AD, Ioio RD, McGowan SJ, Barkoulas M, Galinha C, Rast MI, Hofhuis H, Then C, Plieske J, Ganal M, Mott R, Martinez-Garcia JF, Carine MA, Scotland RW, Gan XC, Filatov DA, Lysak MA, Tsiantis M (2014) Cardamine hirsuta: a versatile genetic system for comparative studies. Plant J 78:1–15. doi: 10.1111/tpj.12447 CrossRefPubMedGoogle Scholar
  14. Herrera-Estrella L, Simpson J, Martinez-Trujillo M (2005) Transgenic plants: an historical perspective. Methods Mol Biol 286:3–32. doi: 10.1385/1-59259-827-7:003 PubMedGoogle Scholar
  15. Higashi H, Ikeda H, Setoguchi H (2012) Population fragmentation causes randomly fixed genotypes in populations of Arabidopsis kamchatica in the Japanese Archipelago. J Plant Res 125:223–233. doi: 10.1007/s10265-011-0436-8 CrossRefPubMedGoogle Scholar
  16. Hu TT, Pattyn P, Bakker EG, Cao J, Cheng JF, Clark RM, Fahlgren N, Fawcett JA, Grimwood J, Gundlach H, Haberer G, Hollister JD, Ossowski S, Ottilar RP, Salamov AA, Schneeberger K, Spannagl M, Wang X, Yang L, Nasrallah ME, Bergelson J, Carrington JC, Gaut BS, Schmutz J, Mayer KF, Van de Peer Y, Grigoriev IV, Nordborg M, Weigel D, Guo YL (2011) The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nat Genet 43:476–481. doi: 10.1038/ng.807 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kuittinen H, Niittyvuopio A, Rinne P, Savolainen O (2008) Natural variation in Arabidopsis lyrata vernalization requirement conferred by a FRIGIDA indel polymorphism. Mol Biol Evol 25:319–329. doi: 10.1093/molbev/msm257 CrossRefPubMedGoogle Scholar
  18. Labra M, Vannini C, Grassi F, Bracale M, Balsemin M, Basso B, Sala F (2004) Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip. Theor Appl Genet 109:1512–1518. doi: 10.1007/s00122-004-1773-y CrossRefPubMedGoogle Scholar
  19. Lackey E, Ng DW, Chen ZJ (2010) RNAi-mediated down-regulation of DCL1 and AGO1 induces developmental changes in resynthesized Arabidopsis allotetraploids. New Phytol 186:207–215. doi: 10.1111/j.1469-8137.2010.03187.x CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lawrence RJ, Pikaard CS (2003) Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. Plant J 36:114–121. doi: 10.1046/j.1365-313X.2003.01857.x CrossRefPubMedGoogle Scholar
  21. Levin DA (2002) The role of chromosomal change in plant evolution. Oxford University Press, New YorkGoogle Scholar
  22. Marchant DB, Soltis DE, Soltis PS (2016) Patterns of abiotic niche shifts in allopolyploids relative to their progenitors. New Phytol 212:708–718. doi: 10.1111/nph.14069 CrossRefGoogle Scholar
  23. Mayrose I, Zhan SH, Rothfels CJ, Magnuson-Ford K, Barker MS, Rieseberg LH, Otto SP (2011) Recently formed polyploid plants diversify at lower rates. Science 333:1257–1257. doi: 10.1126/science.1207205 CrossRefPubMedGoogle Scholar
  24. Mu G, Chang N, Xiang K, Sheng Y, Zhang Z, Pan G (2012) Genetic transformation of maize female inflorescence following floral dip method mediated by Agrobacterium. Biotechnology 11:178–183. doi: 10.3923/biotech.2012.178.183 CrossRefGoogle Scholar
  25. Novikova PY, Hohmann N, Nizhynska V, Tsuchimatsu T, Ali J, Muir G, Guggisberg A, Paape T, Schmid K, Fedorenko OM, Holm S, Sall T, Schlotterer C, Marhold K, Widmer A, Sese J, Shimizu KK, Weigel D, Kramer U, Koch MA, Nordborg M (2016) Sequencing of the genus Arabidopsis identifies a complex history of nonbifurcating speciation and abundant trans-specific polymorphism. Nat Genet 48:1077–1082. doi: 10.1038/ng.3617 CrossRefPubMedGoogle Scholar
  26. Ohno S (1970) Evolution by gene duplication. Springer-Verlag, New YorkGoogle Scholar
  27. Paape T, Hatakeyama M, Shimizu-Inatsugi R, Cereghetti T, Onda Y, Kenta T, Sese J, Shimizu KK (2016) Conserved but attenuated parental gene expression in allopolyploids: constitutive zinc hyperaccumulation in the allotetraploid Arabidopsis kamchatica. Mol Biol Evol 33:2781–2800. doi: 10.1093/molbev/msw141 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Rodriguez RE, Debernardi JM, Palatnik JF (2014) Morphogenesis of simple leaves: regulation of leaf size and shape. Wiley Interdiscip Rev Dev Biol 3:41–57. doi: 10.1002/wdev.115 CrossRefPubMedGoogle Scholar
  29. Schmickl R, Jorgensen MH, Brysting AK, Koch MA (2010) The evolutionary history of the Arabidopsis lyrata complex: a hybrid in the amphi-Beringian area closes a large distribution gap and builds up a genetic barrier. BMC Evol Biol 10:98. doi: 10.1186/1471-2148-10-98 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Schranz ME, Mohammadin S, Edger PP (2012) Ancient whole genome duplications, novelty and diversification: the WGD Radiation Lag-Time Model. Curr Opin Plant Biol 15:147–153. doi: 10.1016/j.pbi.2012.03.011 CrossRefPubMedGoogle Scholar
  31. Shimada TL, Shimada T, Hara-Nishimura I (2010) A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J 61:519–528. doi: 10.1111/j.1365-313X.2009.04060.x CrossRefPubMedGoogle Scholar
  32. Shimizu KK (2002) Ecology meets molecular genetics in Arabidopsis. Popul Ecol 44:221–233. doi: 10.1007/S101440200025 CrossRefGoogle Scholar
  33. Shimizu KK, Tsuchimatsu T (2015) Evolution of selfing: recurrent patterns in molecular adaptation. Annu Rev Ecol Evol Syst 46:593–622. doi: 10.1146/annurev-ecolsys-112414-054249 CrossRefGoogle Scholar
  34. Shimizu K, Fujii S, Marhold K, Watanabe K, Kudoh H (2005) Arabidopsis kamchatica (Fisch. ex DC.) K. Shimizu & Kudoh and A. kamchatica subsp. kawasakiana (Makino) K. Shimizu & Kudoh, new combinations. Acta Phytotax Geobot 56:165–174. doi: 10.18942/apg.KJ00004623241 Google Scholar
  35. Shimizu-Inatsugi R, Lihova J, Iwanaga H, Kudoh H, Marhold K, Savolainen O, Watanabe K, Yakubov VV, Shimizu KK (2009) The allopolyploid Arabidopsis kamchatica originated from multiple individuals of Arabidopsis lyrata and Arabidopsis halleri. Mol Ecol 18:4024–4048. doi: 10.1111/j.1365-294X.2009.04329.x CrossRefPubMedGoogle Scholar
  36. Shimizu-Inatsugi R, Terada A, Hirose K, Kudoh H, Sese J, Shimizu KK (2017) Plant adaptive radiation mediated by polyploid plasticity in transcriptomes. Mol Ecol 26:193–207. doi: 10.1111/mec.13738 CrossRefPubMedGoogle Scholar
  37. Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767. doi: 10.1105/tpc.2.8.755 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Stebbins GL (1971) Chromosomal evolution in higher plants. Edward Arnold (Publishers) Ltd, LondonGoogle Scholar
  39. Sugisaka J, Kudoh H (2008) Breeding system of the annual Cruciferae, Arabidopsis kamchatica subsp. kawasakiana. J Plant Res 121:65–68. doi: 10.1007/s10265-007-0119-7 CrossRefPubMedGoogle Scholar
  40. Trieu AT, Burleigh SH, Kardailsky IV, Maldonado-Mendoza IE, Versaw WK, Blaylock LA, Shin HS, Chiou TJ, Katagi H, Dewbre GR, Weigel D, Harrison MJ (2000) Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 22:531–541. doi: 10.1046/J.1365-313x.2000.00757.X CrossRefPubMedGoogle Scholar
  41. Tsuchimatsu T, Kaiser P, Yew CL, Bachelier JB, Shimizu KK (2012) Recent loss of self-incompatibility by degradation of the male component in allotetraploid Arabidopsis kamchatica. PLoS Genet 8:e1002838. doi: 10.1371/journal.pgen.1002838 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Wood TE, Takebayashi N, Barker MS, Mayrose I, Greenspoon PB, Rieseberg LH (2009) The frequency of polyploid speciation in vascular plants. Proc Natl Acad Sci USA 106:13875–13879. doi: 10.1371/journal.pgen.1002838 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Yasmeen A, Mirza B, Inayatullah S, Safdar N, Jamil M, Ali S, Choudhry MF (2009) In planta transformation of tomato. Plant Mol Biol Rep 27:20–28. doi: 10.1007/s11105-008-0044-5 CrossRefGoogle Scholar
  44. Zale JM, Agarwal S, Loar S, Steber CM (2009) Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens. Plant Cell Rep 28:903–913. doi: 10.1007/s00299-009-0696-0 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Zhang X, Henriques R, Lin SS, Niu QW, Chua NH (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1:641–646. doi: 10.1038/nprot.2006.97 CrossRefPubMedGoogle Scholar
  46. Zozomova-Lihova J, Krak K, Mandakova T, Shimizu KK, Spaniel S, Vit P, Lysak MA (2014) Multiple hybridization events in Cardamine (Brassicaceae) during the last 150 years: revisiting a textbook example of neoallopolyploidy. Ann Bot 113:817–830. doi: 10.1093/aob/mcu012 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan KK 2017

Authors and Affiliations

  • Chow-Lih Yew
    • 1
  • Hiroyuki Kakui
    • 1
    • 2
  • Kentaro K. Shimizu
    • 1
    • 2
    Email author
  1. 1.Department of Evolutionary Biology and Environmental StudiesUniversity of ZurichZurichSwitzerland
  2. 2.Kihara Institute for Biological ResearchYokohama City UniversityYokohamaJapan

Personalised recommendations