Skip to main content
SpringerLink
Log in

Efficient leaf mesophyll protoplast isolation and transient gene expression in bermudagrasses (Cynodon spp.)

  • Research Note
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Bermudagrasses (Cynodon spp.) are important warm-season turfgrass species with multiple applications. Although physiological and high-throughput omics studies were conducted to explore the growth, development and stress responses of bermudagrasses, the molecular mechanism how their turf traits form remain unclear due to difficulties in genetic and transgenic manipulations. In this study, we reported an optimized enzymatic digestion method to stably isolate leaf mesophyll protoplasts from different bermudagrass cultivars. Furthermore, transient expression of two bermudagrass proteins, TEOSINTE BRANCHED1 and phytoene desaturase with different subcellular locations, were successfully observed using the isolated protoplasts. These results suggested that the leaf mesophyll protoplast system could be utilized in gene functional studies of bermudagrasses. As we known, this is the first systematic report of protoplast isolation and application in warm-season turfgrasses, especially Cynodon spp.

Key message:

Leaf mesophyll protoplasts were isolated from eight bermudagrass cultivars and were used to transiently express two bermudagrass genes.

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

Data availability

All data generated or analysed during this study are included in this published article.

References

  • Abel S, Theologis A (1994) Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J 5:421–427

    Article  CAS  Google Scholar 

  • Adedeji OS, Naing AH, Kim CK (2020) Protoplast isolation and shoot regeneration from protoplast-derived calli of Chrysanthemum cv. White ND. Plant Cell Tiss Organ Cult 141:571–581

    Article  Google Scholar 

  • Cha OK, Lee J, Lee HS, Lee H (2019) Optimized protoplast isolation and establishment of transient gene expression system for the Antarctic flowering plant Colobanthus quitensis (Kunth) Bartl. Plant Cell Tiss Organ Cult 138:603–607

    Article  CAS  Google Scholar 

  • Chen J, Zong J, Li D, Chen Y, Wang Y, Guo H, Li J, Li L, Guo S, Liu J (2019) Growth response and ion homeostasis in two bermudagrass (Cynodon dactylon) cultivars differing in salinity tolerance under salinity stress. Soil Sci Plant Nutr 65:419–429

    Article  CAS  Google Scholar 

  • Chen S, Xu X, Ma Z, Liu J, Zhang B (2021) Organ-specific transcriptome analysis identifies candidate genes involved in the stem specialization of bermudagrass (Cynodon dactylon L.). Front Genet 12:678673

    Article  Google Scholar 

  • Figueira TR, Melo DR, Vercesi AE, Castilho RF (2012) Safranine as a fluorescent probe for the evaluation of mitochondrial membrane potential in isolated organelles and permeabilized cells. Methods Mol Biol 810:103–117

    Article  CAS  Google Scholar 

  • Guo Y, Wu Y, Anderson JA, Moss JQ, Zhu L, Fu J (2017) SSR marker development, linkage mapping, and QTL analysis for establishment rate in common bermudagrass. Plant Genome 10:1–11

    Article  Google Scholar 

  • Harlan JR, de Wet JMJ (1969) Sources of variation in Cynodon dactylon (L). Pers Crop Sci 9:774–778

    Article  Google Scholar 

  • Hu Y, Song D, Gao L, Ajayo BS, Wang Y, Huang H, Zhang J, Liu H, Liu Y, Yu G, Liu Y, Li Y, Huang Y (2020) Optimization of isolation and transfection conditions of maize endosperm protoplasts. Plant Methods 16:96

    Article  CAS  Google Scholar 

  • Huang S, Wang C, Liang J (2018) Genetic resources and genetic transformation in bermudagrass – a review. Biotechnol Biotec Eq. 32:1–9

    Article  CAS  Google Scholar 

  • Naing AH, Adedeji OS, Kim CK (2021) Protoplast technology in ornamental plants: current progress and potential applications on genetic improvement. Sci Hortic 283:110043

    Article  CAS  Google Scholar 

  • Niklas KJ, Cobb ED, Matas AJ (2017) The evolution of hydrophobic cell wall biopolymers: from algae to angiosperms. J Exp Bot 68:5261–5269

    Article  CAS  Google Scholar 

  • Page MT, Parry MAJ, Carmo-Silva E (2019) A high-throughput transient expression system for rice. Plant Cell Environ 42:2057–2064

    Article  CAS  Google Scholar 

  • Reasor EH, Brosnan JT, Trigiano RN, Elsner JE, Henry GM, Schwartz BM (2016) The genetic and phenotypic variability of interspecific hybrid bermudagrasses (Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt-Davy) used on golf course putting greens. Planta 244:761–773

    Article  CAS  Google Scholar 

  • Ren R, Gao J, Yin D, Li K, Lu C, Ahmad S, Wei Y, Jin J, Zhu G, Yang F (2021) Highly efficient leaf base protoplast isolation and transient expression systems for orchids and other important monocot crops. Front Plant Sci 12:626015

    Article  Google Scholar 

  • Shao A, Wang W, Fan S, Xu X, Yin Y, Erick A, Li X, Wang G, Wang H, Fu J (2021) Comprehensive transcriptional analysis reveals salt stress-regulated key pathways, hub genes and time-specific responsive gene categories in common bermudagrass (Cynodon dactylon (L.) Pers.) roots. BMC Plant Biol 21:175

    Article  CAS  Google Scholar 

  • Wang J, Wang Y, Lü T, Yang X, Liu J, Dong Y, Wang Y (2022) An efficient and universal protoplast isolation protocol suitable for transient gene expression analysis and single-cell RNA sequencing. Int J Mol Sci 23:3419

    Article  CAS  Google Scholar 

  • Xu X, Liu W, Liu X, Cao Y, Li X, Wang G, Fu C, Fu J (2022) Genetic manipulation of bermudagrass photosynthetic biosynthesis using Agrobacterium-mediated transformation. Physiol Plant 174:e13710

    Article  CAS  Google Scholar 

  • Ye T, Shi H, Wang Y, Yang F, Chan Z (2016) Contrasting proteomic and metabolomic responses of bermudagrass to drought and salt stresses. Front Plant Sci 7:1694

    Article  Google Scholar 

  • Yu G, Cheng Q, Xie Z, Xu B, Huang B, Zhao B (2017) An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1. Plant Methods 13:46

    Article  Google Scholar 

  • Zhang B, Shi JA, Chen JB, Li DD, Li JJ, Guo HL, Zong JQ, Wang Y, Guo AG, Liu JX (2016) Efficient virus-induced gene silencing in Cynodon dactylon and Zoysia japonica using rice tungro bacilliform virus vectors. Sci Hortic 207:97–103

    Article  CAS  Google Scholar 

  • Zhang B, Liu J (2018) Molecular cloning and sequence variance analysis of the TEOSINTE BRANCHED1 (TB1) gene in bermudagrass [Cynodon dactylon (L.) Pers]. J Plant Physiol 229:142–150

    Article  CAS  Google Scholar 

  • Zhao Y, Du H, Wang Z, Huang B (2011) Identification of proteins associated with water-deficit tolerance in C4 perennial grass species, Cynodon dactylon × Cynodon transvaalensis and Cynodon dactylon. Physiol Plant 141:40–55

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (32072613). The funding body has no roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 32072613).

Author information

Authors and Affiliations

  1. College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China

    Si Chen & Bing Zhang

  2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China

    Jianxiu Liu

Authors
  1. Si Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianxiu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SC conducted the research, performed the experiments and drafted the manuscript. BZ conceived the research, designed the experiments and revised the manuscript. JL provided the plant material and helped to write the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Bing Zhang.

Ethics declarations

Conflict of interest

The authors have no conflict of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Additional information

Communicated by Ewa Grzebelus.

Publisher’s Note

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

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 14.4 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, S., Liu, J. & Zhang, B. Efficient leaf mesophyll protoplast isolation and transient gene expression in bermudagrasses (Cynodon spp.). Plant Cell Tiss Organ Cult 152, 229–235 (2023). https://doi.org/10.1007/s11240-022-02394-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-022-02394-2

Keywords

Search

Navigation