Skip to main content
SpringerLink
Log in

Establishment of an efficient Agrobacterium-mediated genetic transformation system in halophyte Puccinellia tenuiflora

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

Alkaligrass (Puccinellia tenuiflora) is a monocotyledonous halophyte pasture, which has strong tolerance to saline-alkali, drought, and chilling stresses. We have reported a high-quality chromosome-level genome and stress-responsive proteomic results in P. tenuiflora. However, the gene/protein function investigations are still lacking, due to the absent of genetic transformation system in P. tenuiflora. In this study, we established a higher efficient Agrobacterium-mediated transformation for P. tenuiflora using calluses induced from seeds. Agrobacterium strain EHA105 harbors pANIC 6B vectors that contain GUS reporter gene and Hyg gene for screening. Ten mg·L−1 hygromycin was used for selecting transgenic calluses. The optimized condition of vacuum for 10 min, ultrasonication for 10 min, and then vacuum for 10 min was used for improvement of conversion efficiency. Besides, 300 mg·L−1 timentin was the optimum antibiotics in transformation. PCR amplification exhibited that GUS gene has been successfully integrated into the chromosome of P. tenuiflora. Histochemical GUS staining and qRT-PCR analysis indicated that GUS gene has stably expressed with ß-glucuronidase activity in transgene seedlings. All these demonstrated that we have successfully established an Agrobacterium-mediated transformation system of P. tenuiflora, which provides a good platform for further gene function analysis and lays a solid foundation for molecular breeding.

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

Similar content being viewed by others

Data availability

Datasets supporting the conclusions of this article are included within the article.

References

  • Alves S, Worland B, Thole V, Thole V, Snape J, Bevan M, Vain P (2009) A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21. Nat Protoc 4:638–649

    Article  CAS  Google Scholar 

  • Bu YY, Zhao MQ, Sun B, Zhang XX, Takano T, Liu SK (2014) An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora. BMC Biotechnol 14:52

    Article  Google Scholar 

  • Esmaeili S, Salehi H, Morteza K, Niazi A, Tohidfar M, Aram F (2019) Isopentenyl transferase (IPT) gene transfer to perennial ryegrass through sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum and heat treatment. Mol Biotechnol 61:332–344

    Article  CAS  Google Scholar 

  • Han Y, Kim Y, Lee J, Kim S, Cho K, Thummala C, Song P, Woo Y, Kim J (2009) Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation. Plant Cell Rep 28:397–406

    Article  Google Scholar 

  • Jefferson R, Kavanagh T, Bevan M (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    Article  CAS  Google Scholar 

  • Jongjitvimol T, Sujipuli K, Urtgam S (2020) Application of sonication-and vacuum infiltration-assisted Agrobacterium-mediated transformation of rice embryo (Oryza sativa L.). ScienceAsia 46:412–419

    Article  CAS  Google Scholar 

  • Kang JN, Park MY, Kim WN, Kang HG, Sun HJ, Yang DH, Ko SM, Lee HY (2017) Resistance of transgenic zoysiagrass overexpressing the zoysiagrass class II chitinase gene Zjchi2 against Rhizoctonia solani AG2-2 (IV). Plant Biotechnol Rep 11:229–238

    Article  Google Scholar 

  • Kim SJ, Lee JY, Kim YM, Yang SS, Hwang OJ, Hong NJ, Kim KM, Lee HY, Son PS, Kim JI (2007) Agrobacterium-mediated high-efficiency transformation of creeping bentgrass with herbicide resistance. J Plant Biol 50:577–585

    Article  CAS  Google Scholar 

  • Kumar J, Shukla SM, Bhat V, Gupta S, Gupta MG (2005) In vitro plant regeneration and genetic transformation of Dichanthium annulatum. DNA Cell Bio 24:670–679

    Article  CAS  Google Scholar 

  • Li RY, Qu RD (2011) High throughput Agrobacterium-mediated switchgrass transformation. Biomass Bioenerg 35:1046–1054

    Article  CAS  Google Scholar 

  • Liu MX, Lu SY, Liu L, Tan JL, Guo ZF (2012) Agrobacterium-mediated transformation of centipedegrass (Eremochloa ophiuroides [Munro] Hack.). Plant Cell Tiss Org 109:557–563

    Article  CAS  Google Scholar 

  • Liu YR, Cen HF, Yan JP, Zhang YW, Zhang WJ (2015) Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars. Plant Cell Rep 34:1099–1108

    Article  CAS  Google Scholar 

  • Mann D, Lafayette P, Abercrombie L, King Z, Mazarei M, Halter M, Poovaiah C, Baxter H, Shen H, Dixon R, Parrott W, Neal S (2012) Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species. Plant Biotechnol J 10:226–236

    Article  CAS  Google Scholar 

  • Meng XJ, Zhao Q, Jin YD, Yu JJ, Yin ZP, Chen SX, Dai SJ (2016) Chilling-responsive mechanisms in halophyte Puccinellia tenuiflora seedlings revealed from proteomics analysis. J Proteomics 143:365–381

    Article  CAS  Google Scholar 

  • Nataliya K, Wei J, Omid E, Sarah M, Tatiana P, Stephen F, Natalia B, John H, Kontanze B, Yuri S, Peter L, Sergiy L (2013) Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters. Plant Biotechnol J 11:659–670

    Article  Google Scholar 

  • Sieburth L, Meyerowitz E (1997) Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9:355–365

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sinicae Agendae Academiae Sinicae Edita (2002) Flora reipublicae popularis sinicae. Science Press, Beijing, p 236

    Google Scholar 

  • Soyza R, Alves M, Carneiro N (2017) Agrobacterium-mediated genetic transformation of a tropical elite maize line. Crop Breed Appl Biot 17:133–140

    Article  Google Scholar 

  • Sun YL, Hong SK (2012) Agrobacterium tumefaciens-mediated transformation of the halophyte Leymus chinensis (Trin.). Plant Mol Biol Rep 30:1253–1263

    Article  CAS  Google Scholar 

  • Suo JW, Zhang H, Zhao Q, Zhang N, Zhang YX, Li Y, Song BH, Yu JJ, Cao JG, Wang T, Luo J, Guo LH, Ma J, Zhang XM, She YM, Peng LW, Ma WM, Guo SY, Miao YC, Chen SX, Qin Z, Dai SJ (2020) Na2CO3-responsive photosynthetic and ROS scavenging mechanisms in chloroplasts of alkaligrass revealed by phosphoproteomics. Genom Proteom Bioinf 18:271–288

    Article  Google Scholar 

  • Takamizo T, Sato H (2020) Protocol for Agrobacterium-mediated transformation of tall fescue and future perspective on the application of genome editing. Plant Biotechnology 37:157–161

    Article  CAS  Google Scholar 

  • Tanweer K, Uzma MR, Zaheer A, Ghulam MA (2014) Genetic improvement of sugarcane for drought and salinity stress tolerance using Arabidopsis vacuolar pyrophosphatase (AVP1). Gene Mol Biotechnol 56:199–209

    Article  Google Scholar 

  • Touchkanin J, Kawee S, Sittichai U (2020) Application of sonication- and vacuum infiltration-assisted Agrobacterium-mediated transformation of rice embryo (Oryza sativa L.). ScienceAsia 46:412–419

    Article  Google Scholar 

  • Wang F, Jiang J (2014) Effects of different antibiotics on induction of Callus from the leaves of emopenBetula pendulaemclose ‘Purple Rain.’ J Southwest For Univ Nat Sci 34:27–31. (In Chinese with English abstract)

    Google Scholar 

  • Wang K, Gong Q, Ye XG (2020) Recent developments and applications of genetic transformation and genome editing technologies in wheat. Theor Appl Genet 133:1603–1622

    Article  CAS  Google Scholar 

  • Wang T, Han X, Zhao MQ, Takano T, Liu SK (2011) Studies on construction of regeneration system and genetic transformation of Puccinellia chinampoensis. Bioscience Methods. https://doi.org/10.5376/bm2011.02.0005

    Article  Google Scholar 

  • Wang YC, Chu YG, Liu GF, Wang MH, Jiang J, Hou YJ, Qu GZ, Yang CP (2007) Identification of expressed sequence tags in an alkali grass (Puccinellia tenuiflora) cDNA library. J Plant Physiol 164:78–89

    Article  CAS  Google Scholar 

  • Wu E, Lenderts B, Glassman K, Berezowska K, Christensen H, Asmus T, Zhen SF, Chu U, Cho M, Zhao ZY (2014) Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants. Vitro Cell Dev-Pl 50:9–18

    Article  Google Scholar 

  • Yan XF, Sun GR (2000) Physiological ecology research of Puccinellia tenuiflora. Science Press, Beijing

    Google Scholar 

  • Yin ZP, Zhang H, Zhao Q, Yoo MJ, Zhu N, Yu JL, Yu JJ, Guo SY, Miao YC, Chen SX, Qin Z, Dai SJ (2019) Physiological and comparative proteomic analyses of saline-alkali NaHCO3-responses in leaves of halophyte Puccinellia tenuiflora. Plant Soil 437:137–158

    Article  CAS  Google Scholar 

  • Yu JJ, Chen SX, Wang T, Sun GR, Dai SJ (2013) Comparative proteomic analysis of Puccinellia tenuiflora leaves under Na2CO3 stress. Int J Mol Sci 14:1740–1762

    Article  CAS  Google Scholar 

  • Yu JJ, Chen SX, Zhao Q, Wang T, Yang CP, Diaz C, Sun GR, Dai SJ (2011) Physiological and proteomic analysis of salinity tolerance in Puccinellia tenuiflora. J Proteome Res 10:3852–3870

    Article  CAS  Google Scholar 

  • Yu JJ, Zhang YX, Liu JM, Wang L, Liu PP, Yin ZP, Guo SY, Ma J, Lu Z, Wang T, She YM, Miao YC, Ma L, Chen SX, Li Y, Dai SJ (2018) Proteomic discovery of H2O2 response in roots and functional characterization of PutGLP gene from alkaligrass. Planta 248:1079–1099

    Article  CAS  Google Scholar 

  • Zhang WT, Liu J, Zhang YX, Qiu J, Li Y, Zheng BJ, Hu FH, Dai SJ, Huang XH (2020) A high-quality genome sequence of alkaligrass provides insights into halophyte stress tolerance. Sci China Life Sci 63:1269–1282

    Article  CAS  Google Scholar 

  • Zhang X, Wei LQ, Wang ZZ, Wang T (2013) Physiological and molecular features of Puccinellia tenuiflora tolerating salt and alkaline-salt stress. J Integr Plant Biol 55:262–276

    Article  CAS  Google Scholar 

  • Zhang YX, Zhang Y, Yu JJ, Zhang H, Wang LY, Wang SN, Guo SY, Miao YC, Chen SX, Li Y, Dai SJ (2019) NaCl-responsive ROS scavenging and energy supply in alkaligrass callus revealed from proteomic analysis. BMC Genomics 20:990

    Article  CAS  Google Scholar 

  • Zhao Q, Suo JW, Chen SX, Jin YD, Ma XL, Yin ZP, Zhang YH, Wang T, Luo J, Jin WH, Zhang X, Zhou ZQ, Dai SJ (2016) Na2CO3-responsive mechanisms in halophyte Puccinellia tenuiflora roots revealed by physiological and proteomic analyses. Sci Rep-UK 6:32717

    Article  CAS  Google Scholar 

Download references

Funding

This study was supported by the National Natural Science Foundation of China (No. 32070300), the Fund of Central Government Guides Local Science and Technology Development, China (No. YDZX20203100003927), and the Fund of Shanghai Engineering Research Center of Plant Germplasm Resources, China (No.17DZ2252700) to S. D.

Author information

Authors and Affiliations

  1. Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai, 200234, China

    Yue Zhang, Chunxiao Qin, Shijia Liu, Yongxue Zhang, Yingying Song, Meihong Sun, Zhi Qin & Shaojun Dai

  2. Key Laboratory of Saline-Alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, College of Life Sciences, Northeast Forestry University, Harbin, 150040, China

    Yue Zhang, Chunxiao Qin, Shijia Liu, Ying Li & Yingying Song

  3. Shandong Technology Innovation Center of Synthetic Biology, Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266000, China

    Yue Xu & Chunxiang Fu

Authors
  1. Yue Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chunxiao Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shijia Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ying Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yongxue Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yingying Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Meihong Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chunxiang Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zhi Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Shaojun Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Project design: SJ. D., Z. Q., MH. S., and CX. F.; experimental work, Y. Z., CX. Q., SJ. L., and YY. S.; data analysis, Y. X., YX. Z., MH. S., Z. Q., and Y. L.; writing manuscript, Y. Z., Z. Q., CX. F., and SJ. D. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Zhi Qin or Shaojun Dai.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

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 file1 (DOC 31.0 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, Y., Qin, C., Liu, S. et al. Establishment of an efficient Agrobacterium-mediated genetic transformation system in halophyte Puccinellia tenuiflora. Mol Breeding 41, 55 (2021). https://doi.org/10.1007/s11032-021-01247-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11032-021-01247-8

Keywords

Search

Navigation