Skip to main content
SpringerLink
Log in

Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae)

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

This is the first report of a highly efficient Agrobacterium tumefaciens-mediated transformation protocol for Acanthaceae and its utilization in revealing important roles of cytokinin in regulating heterophylly in Hygrophila difformis.

Abstract

Plants show amazing morphological differences in leaf form in response to changes in the surrounding environment, which is a phenomenon called heterophylly. Previous studies have shown that the aquatic plant Hygrophila difformis (Acanthaceae) is an ideal model for heterophylly study. However, low efficiency and poor reproducibility of genetic transformation restricted H. difformis as a model plant. In this study, we reported successful induction of callus, shoots and the establishment of an efficient stable transformation protocol as mediated by Agrobacterium tumefaciens LBA4404. We found that the highest callus induction efficiency was achieved with 1 mg/L 1-Naphthaleneacetic acid (NAA) and 2 mg/L 6-benzyladenine (6-BA), that efficient shoot induction required 0.1 mg/L NAA and 0.1 mg/L 6-BA and that high transformation efficiency required 100 µM acetosyringone. Due to the importance of phytohormones in the regulation of heterophylly and the inadequate knowledge about the function of cytokinin (CK) in this process, we analyzed the function of CK in the regulation of heterophylly by exogenous CK application and endogenous CK detection. By using our newly developed transformation system to detect CK signals, contents and distribution in H. difformis, we revealed an important role of CK in environmental mediated heterophylly.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Aloni R, Aloni E, Langhans M, Ullrich CI (2006) Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism. Ann Bot 97:883–893

    CAS  PubMed  PubMed Central  Google Scholar 

  • Baker-Brosh KF, Peet RK (1997) The ecological significance of lobed and toothed leaves in temperature forest trees. Ecology 78:1250–1255

    Google Scholar 

  • Bhawna M, Kumar S, Kumar R (2017) Pharmacognostical studies of in vivo grown garden plant and in vitro generated plantlets from nodal explants of Justicia adhatoda L. Int J Bioassays 6:5230–5235

    CAS  Google Scholar 

  • Bristow JM (1969) The effects of carbon dioxide on the growth and development of amphibious plants. Can J Bot 47:1803–1807

    CAS  Google Scholar 

  • Bristow JM, Looi AS (1968) Effects of carbon dioxide on the growth and morphogenesis of Marsilea. Am J Bot 55:884–889

    CAS  Google Scholar 

  • Chhabra G, Chaudhary D, Sainger M, Jaiwal PK (2011) Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants. Physiol Mol Biol Plants 17:129–136

    CAS  PubMed  PubMed Central  Google Scholar 

  • Clarke JD (2009) Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb Protoc 3:5177

    Google Scholar 

  • Cook CDK (1969) On the determination of leaf form in Ranunculus aquatilis. New Phytol 68:469–480

    Google Scholar 

  • Deschamp PA, Cooke TJ (1984) Causal mechanisms of leaf dimorphism in the aquatic angiosperm Callitriche heterophylla. Am J Bot 71:319–329

    Google Scholar 

  • Drisyadas P, Smitha RB, Benjamin S, Madhusoodanan PV (2014) Induction of in-vitro flowering and callogenesis in Blepharis maderaspatensis L. (Acanthaceae), a medicinal plant. Ann Plant Sci 3:799–803

    Google Scholar 

  • Eckardt NA (2005) Cross talk between gibberellin and cytokinin signaling converges on SPINDLY. Plant Cell 17:1–3

    CAS  PubMed Central  Google Scholar 

  • Efroni I, Han SK, Kim HJ, Wu MF, Steiner E, Birnbaum K, Hong JC, Eshed Y, Wagner D (2013) Regulation of leaf maturation by chromatin-mediated modulation of cytokinin responses. Dev Cell 24:438–445

    CAS  PubMed  PubMed Central  Google Scholar 

  • Farber M, Attia Z, Weiss D (2016) Cytokinin activity increases stomatal density and transpiration rate in tomato. J Exp Bot 67:6351–6362

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gan Y, Liu C, Broun HP (2007) Integration of cytokinin and gibberellin signalling by Arabidopsis transcription factors GIS, ZFP8 and GIS2 in the regulation of epidermal cell fate. Development 134:2073–2081

    CAS  PubMed  Google Scholar 

  • He D, Guo P, Gugger PF (2018) Investigating the molecular basis for heterophylly in the aquatic plant Potamogeton octandrus (Potamogetonaceae) with comparative transcriptomics. Peerj 6:e4448

    PubMed  PubMed Central  Google Scholar 

  • Helliker BR, Richter SL (2008) Subtropical to boreal convergence of tree-leaf temperatures. Nature 454:511

    CAS  PubMed  Google Scholar 

  • Holst K, Schmülling T, Werner T (2011) Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation. J Plant Physiol 168:1328–1334

    CAS  PubMed  Google Scholar 

  • Horiguchi G, Nemoto K, Yokoyama T, Hirotsu N (2019) Photosynthetic acclimation of terrestrial and submerged leaves in the amphibious plant Hygrophila difformis. AoB Plants 11:plz009

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ikeuchi M, Sugimoto K, Iwase A (2013) Plant callus: mechanisms of induction and repression. Plant Cell 25:3159–3173

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jackson MB (2007) Ethylene-promoted elongation: an adaptation to submergence stress. Ann Bot 101:229–248

    PubMed  PubMed Central  Google Scholar 

  • Johnson MP (1967) Temperature dependent leaf morphogenesis in Ranunculus flabellaris. Nature 214:1354–1355

    Google Scholar 

  • Kim J, Joo Y, Kyung J, Jeon M, Park J, Lee HG, Chung DS, Lee EJ, Lee I (2018) A molecular basis behind heterophylly in an amphibious plant Ranunculus trichophyllus. PLoS Genet 14(2):e1007208

    PubMed  PubMed Central  Google Scholar 

  • Kuwabara A, Ikegami K, Koshiba T, Nagata T (2003) Effects of ethylene and abscisic acid upon heterophylly in Ludwigia arcuata (Onagraceae). Planta 217:880–887

    CAS  PubMed  Google Scholar 

  • Kuwabara A, Tsukaya H, Nagata T (2001) Identification of factors that cause heterophylly in Ludwigia arcuata Walt. (Onagraceae). Plant Biol 3:98–105

    Google Scholar 

  • Li G, Hu S, Yang J, Schultz EA, Clarke K, Hou H (2017) Water-Wisteria as an ideal plant to study heterophylly in higher aquatic plants. Plant Cell Rep 36:1–12

    CAS  Google Scholar 

  • Li L, Shao T, Yang H, Chen M, Gao X, Long X, Shao H, Liu Z, Rengel Z (2016) The endogenous plant hormones and ratios regulate sugar and dry matter accumulation in Jerusalem artichoke in salt-soil. Sci Total Environ 578:40

    PubMed  Google Scholar 

  • Li G, Hu S, Hou H, Kimura S (2019a) Heterophylly: phenotypic plasticity of leaf shape in aquatic and amphibious plants. Plants 8:420

    CAS  PubMed Central  Google Scholar 

  • Li X, He D, Guo Y (2019b) Morphological structure and physiological research of heterophylly in Potamogeton octandrus. Plant Syst Evol 305:223–232

    Google Scholar 

  • Lin BL, Yang WJ (1999) Blue light and abscisic acid independently induce heterophyllous switch in Marsilea quadrifolia. Plant Physiol 119:429–434

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mariashibu TS, Subramanyam K, Arun M, Mayavan S, Rajesh M, Theboral J, Manickavasagam M, Ganapathi A (2013) Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars. Acta Physiol Plant 35:41–54

    CAS  Google Scholar 

  • Müller B, Sheen J (2008) Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453:1094–1097

    PubMed  PubMed Central  Google Scholar 

  • Nakayama H, Nakayama N, Seiki S, Kojima M, Sinha N, Kimura S (2014) Regulation of the KNOX-GA gene module induces heterophyllic alteration in North American Lake Cress. Plant Cell 26(12):4733–4748

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nakayama H, Sinha N, Kimura S (2017) How do plants and phytohormones accomplish heterophylly, leaf phenotypic plasticity, in response to environmental cues. Front in Plant Sci 8:1717

    Google Scholar 

  • Ogaki M, Furuichi Y, Kuroda K, Chin DP, Ogawa Y, Mii M (2008) Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium x formolongi. Plant Cell Rep 27:699–705

    CAS  PubMed  Google Scholar 

  • Panigrahi J, Behera M, Maharana S, Mishra RR (2007) Biomolecular changes during in vitro organogenesis of Asteracantha longifolia (L.) Nees—a medicinal herb. Indian J Exp Biol 45:911–919

    CAS  PubMed  Google Scholar 

  • Rascio N, Cuccato F, Vecchia FD, La Rocca N, Larcher W (1999) Structural and functional features of the leaves of Ranunculus trichophyllus Chaix. a freshwater submerged macrophophyte. Plant, Cell Environ 22:205–212

    Google Scholar 

  • Sack L, Scoffoni C, Mckown AD, Frole K, Rawls M, Havran JC, Tran HA, Tran T (2012) Developmentally based scaling of leaf venation architecture explains global ecological patterns. Nat Commun 3:837

    PubMed  Google Scholar 

  • Shani E, Ben-Gera H, Shleizer-Burko S, Burko Y, Ori WN (2010) Cytokinin regulates compound leaf development in tomato. Plant Cell 22(10):3206–3217

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shanks CM, Hecker A, Cheng CI, Brand L, Kieber JJ (2018) Role of BASIC PENTACYSTEINE transcription factors in a subset of cytokinin signaling responses. Plant J 95(3):458–473

    CAS  PubMed  Google Scholar 

  • Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298

    CAS  PubMed  Google Scholar 

  • Singh RK, Prasad M (2016) Advances in Agrobacterium tumefaciens -mediated genetic transformation of graminaceous crops. Protoplasma 253:691–707

    CAS  PubMed  Google Scholar 

  • Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. In: Symposia of the Society for experimental biology, pp 118–131.

  • Su YH, Liu YB, Zhang XS (2011) Auxin-cytokinin interaction regulates meristem development. Mol Plant 4:616–625

    CAS  PubMed  PubMed Central  Google Scholar 

  • Takatsuka H, Umeda M (2019) ABA inhibits root cell elongation through repressing the cytokinin signaling. Plant Signal Behav 14:e1578632

    PubMed  PubMed Central  Google Scholar 

  • Titus JE, Gary Sullivan P (2001) Heterophylly in the yellow waterlily, Nuphar variegata (Nymphaeaceae): effects of [CO2], natural sediment type, and water depth. Am J Bot 88:1469–1478

    CAS  PubMed  Google Scholar 

  • Trieu AT, Burleigh SH, Kardailsky IV, Maldonado Mendoza IE, Versaw WK, Blaylock LA, Heungsop S (2000) Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 22:531–541

    CAS  PubMed  Google Scholar 

  • Varshney A, Shahzad A, Anis M (2009) High frequency induction of somatic embryos and plantlet regeneration from nodal explants of Hygrophila spinosa T. Anders. Afr J Biotechnol 8:6141–6145

    CAS  Google Scholar 

  • Wanke D (2011) The ABA-mediated switch between submersed and emersed life-styles in aquatic macrophytes. J Plant Res 124:467–475

    CAS  PubMed  Google Scholar 

  • Wells CL, Pigliucci M (2000) Adaptive phenotypic plasticity: the case of heterophylly in aquatic plants. Perspect Plant Ecol Evol Syst 3:1–18

    Google Scholar 

  • Werner T, Motyka V, Laucou V, Smets R, Onckelen HV, Schmulling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xi J, Patel M, Dong S, Que Q, Qu R (2018) Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus. BMC Biotechnol 18:48

    PubMed  PubMed Central  Google Scholar 

  • Yang J, Li G, Hu S, Bishopp A, Heenatigala P, Kumar S, Duan P, Yao L, Hou H (2018) A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens. Aquat Bot 151:80–86

    Google Scholar 

  • Zotz G, Wilhelm K, Becker A (2011) Heteroblasty—a review. Bot Rev 77:109–151

    Google Scholar 

Download references

Acknowledgments

This work was supported by grants to Prof. Hongwei Hou from Project of State Key Laboratory of Freshwater Ecology and Biotechnology (2019FB11), Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences (AB2018020), National Key R & D Program (2017YFE0128800, 2018YFD0900801) and General Project of Natural Science Foundation (31870384). This work was also supported by JSPS KAKENHI (JP16H01472, JP16K07408, 18H04787 and 18H04844 to S.K.) and by the MEXT Supported Program for the Strategic Research Foundation at Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant Number S1511023 to S.K. This work was also supported by NSERC Discovery Grant (RGPIN-2017-04381 to Elizabeth A. Schultz).

Author information

Author notes

  1. Gaojie Li, Shiqi Hu contributed equally to this work.

Authors and Affiliations

  1. The State Key Laboratory of Freshwater Ecology and Biotechnology, The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, Hubei, People’s Republic of China

    Gaojie Li, Shiqi Hu, Jingjing Yang, Xuyao Zhao & Hongwei Hou

  2. University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Gaojie Li, Shiqi Hu, Jingjing Yang, Xuyao Zhao & Hongwei Hou

  3. Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto-shi, Kyoto, 603-8555, Japan

    Seisuke Kimura

  4. Center for Ecological Evolutionary Developmental Biology, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto-shi, Kyoto, 603-8555, Japan

    Seisuke Kimura

  5. Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, Canada

    Elizabeth A. Schultz

Authors
  1. Gaojie Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shiqi Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jingjing Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xuyao Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Seisuke Kimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elizabeth A. Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hongwei Hou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HH planned and designed the research. GL, SH and JY performed experiments and data analysis. GL, SH, XZ, SK, EAS, HH contributed to the writing and editing of this article.

Corresponding author

Correspondence to Hongwei Hou.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by Xian Sheng Zhang.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 16 kb)

Fig. S1. The structure of TCS::GUS construct for transformation of H. difformis

299_2020_2527_MOESM3_ESM.tif

Fig. S2. Plant and leaf morphology upon control and lovastatin treatment.(A) Top view of plants grown in 26 °C, 60% RH conditions. Note that plants grown in this condition and treated with 0.1% (w/v) ethanol was set as control.(B) Top view of plants treated with 5 µM lovastatin.(C) Leaf shape of control.(D) Leaf shape of plants treated with 5 µM lovastatin.Bars =1 cm in (A)-(D)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, G., Hu, S., Yang, J. et al. Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae). Plant Cell Rep 39, 737–750 (2020). https://doi.org/10.1007/s00299-020-02527-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-020-02527-x

Keywords

Search

Navigation