An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus

Abstract

Rapeseed (Brassica napus) is an important oil crop that supplies a considerable amount of global vegetable oil production. Genetic transformation system is important to gene functional analysis and molecular breeding. Here, an efficient Agrobacterium-mediated transformation protocol using hypocotyl of rapeseed as explants is described. To develop this protocol, we compared several essential factors that would affect the transformation efficiency, such as Agrobacterium strains, selection marker genes, and genotypes of rapeseed. Comparison of different Agrobacterium strains showed that the GV3101 had higher transformation efficiency than that of C58C1 and EHA105. HPTII, NPTII, and RePAT were used as selection marker genes in tissue culture. The results showed that the transformation efficiency was 3.7–4.8%, 2.2–22.5%, and 1.6–5.9% when the hypocotyl of Westar was infected by GV3101 and screened under hygromycin, kanamycin, and basta, respectively. The transformation efficiency of Westar was the highest and ZS11 was the lowest when five different genotypes of rapeseed (Westar, ZS9, ZS11, GY284, and WH3417) were infected by GV3101. Using this protocol, it will take 8–10 weeks to obtain transgenic plants. This protocol has been used to study gene function in several genotypes of rapeseed in our laboratory. These results indicate that it is efficient to obtain transgenic plant of rapeseed using this protocol.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Berg DE, Davies J, Allet B, Rochaix JD (1975) Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci U S A 72(9):3628–3632. https://doi.org/10.1073/pnas.72.9.3628

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Bergman P, Glimelius K (1993) Electroporation of rapeseed protoplasts – transient and stable transformation. 88(4):604–611. https://doi.org/10.1111/j.1399-3054.1993.tb01378.x

  3. Bhalla PL, Singh MB (2008) Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea. Nat Protoc 3(2):181–189. https://doi.org/10.1038/nprot.2007.527

    CAS  Article  PubMed  Google Scholar 

  4. Chalhoub B, Denoeud F, Liu S, Parkin IA, Tang H, Wang X, Chiquet J, Belcram H, Tong C, Samans B, Correa M, Da Silva C, Just J, Falentin C, Koh CS, Le Clainche I, Bernard M, Bento P, Noel B, Labadie K, Alberti A, Charles M, Arnaud D, Guo H, Daviaud C, Alamery S, Jabbari K, Zhao M, Edger PP, Chelaifa H, Tack D, Lassalle G, Mestiri I, Schnel N, Le Paslier MC, Fan G, Renault V, Bayer PE, Golicz AA, Manoli S, Lee TH, Thi VH, Chalabi S, Hu Q, Fan C, Tollenaere R, Lu Y, Battail C, Shen J, Sidebottom CH, Wang X, Canaguier A, Chauveau A, Berard A, Deniot G, Guan M, Liu Z, Sun F, Lim YP, Lyons E, Town CD, Bancroft I, Wang X, Meng J, Ma J, Pires JC, King GJ, Brunel D, Delourme R, Renard M, Aury JM, Adams KL, Batley J, Snowdon RJ, Tost J, Edwards D, Zhou Y, Hua W, Sharpe AG, Paterson AH, Guan C, Wincker P (2014) Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345(6199):950–953. https://doi.org/10.1126/science.1253435

    CAS  Article  PubMed  Google Scholar 

  5. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x

    CAS  Article  PubMed  Google Scholar 

  6. Cui Y, Liu Z, Li Y, Zhou F, Chen H, Lin Y (2016) Application of a novel phosphinothricin N-acetyltransferase (RePAT) gene in developing glufosinate-resistant rice. Sci Rep 6:21259–21259. https://doi.org/10.1038/srep21259

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133(2):462–469. https://doi.org/10.1104/pp.103.027979

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23(2):131–171. https://doi.org/10.1016/j.biotechadv.2004.09.008

    CAS  Article  PubMed  Google Scholar 

  9. Davey MR, Soneji JR, Rao MN, Kourmpetli S, Bhattacharya A, Kole C (2010) Generation and deployment of transgenic crop plants: an overview. In: Kole C, Michler CH, Abbott AG, Hall TC (eds) Transgenic crop plants: principles and development. Springer, Berlin Heidelberg, pp 1–29. https://doi.org/10.1007/978-3-642-04809-8_1

    Google Scholar 

  10. Donn G, Köcher H (2002) Inhibitors of glutamine synthetase. In pp 87–101. https://doi.org/10.1007/978-3-642-59416-8_4

  11. Eimert K, Siegemund F (1992) Transformation of cauliflower (Brassica oleracea L. var. botrytis)--an experimental survey. Plant Mol Biol 19(3):485–490. https://doi.org/10.1007/BF00023396

    CAS  Article  PubMed  Google Scholar 

  12. Gracka A, Jelen HH, Majcher M, Siger A, Kaczmarek A (2016) Flavoromics approach in monitoring changes in volatile compounds of virgin rapeseed oil caused by seed roasting. J Chromatogr A 1428:292–304. https://doi.org/10.1016/j.chroma.2015.10.088

    CAS  Article  PubMed  Google Scholar 

  13. Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25(2–3):179–188. https://doi.org/10.1016/0378-1119(83)90223-8

    CAS  Article  PubMed  Google Scholar 

  14. Hansen LN, Ortiz R, Andersen SB (1999) Genetic analysis of protoplast regeneration ability in Brassica oleracea. Plant Cell Tissue Organ Cult 58(2):127–132. https://doi.org/10.1023/A:1006359804328

    Article  Google Scholar 

  15. Herrera-Estrella L, Simpson J, Martínez-Trujillo M (2004) Transgenic plants: an historical perspective. In: Peña L (ed) Transgenic plants: methods and protocols. Humana Press, Totowa, pp 3–31. https://doi.org/10.1385/1-59259-827-7:003

    Google Scholar 

  16. Hong Y, Pan X, Welti R, Wang X (2008) Phospholipase Dalpha3 is involved in the hyperosmotic response in Arabidopsis. Plant Cell 20(3):803–816. https://doi.org/10.1105/tpc.107.056390

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Ishida Y, Hiei Y, Komari T (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2(7):1614–1621. https://doi.org/10.1038/nprot.2007.241

    CAS  Article  PubMed  Google Scholar 

  18. Kikkert JR, Vidal JR, Reisch BI (2005) Stable transformation of plant cells by particle bombardment/biolistics. Methods Mol Biol 286:61–78. https://doi.org/10.1385/1-59259-827-7:061

    CAS  Article  PubMed  Google Scholar 

  19. Lu S, Bahn SC, Qu G, Qin H, Hong Y, Xu Q, Zhou Y, Hong Y, Wang X (2013) Increased expression of phospholipase Dalpha1 in guard cells decreases water loss with improved seed production under drought in Brassica napus. Plant Biotechnol J 11(3):380–389. https://doi.org/10.1111/pbi.12028

    CAS  Article  PubMed  Google Scholar 

  20. Lu S, Fadlalla T, Tang S, Li L, Ali U, Li Q, Guo L (2019) Genome-wide analysis of phospholipase D gene family and profiling of phospholipids under abiotic stresses in Brassica napus. Plant Cell Physiol 60:1556–1566. https://doi.org/10.1093/pcp/pcz071

    CAS  Article  PubMed  Google Scholar 

  21. Lu S, Yao S, Wang G, Guo L, Zhou Y, Hong Y, Wang X (2016) Phospholipase Depsilon enhances Braasca napus growth and seed production in response to nitrogen availability. Plant Biotechnol J 14(3):926–937. https://doi.org/10.1111/pbi.12446

    CAS  Article  PubMed  Google Scholar 

  22. Mason AS, Snowdon RJ (2016) Oilseed rape: learning about ancient and recent polyploid evolution from a recent crop species. Plant Biol (Stuttg) 18(6):883–892. https://doi.org/10.1111/plb.12462

    CAS  Article  Google Scholar 

  23. Mukhopadhyay A, Topfer R, Pradhan AK, Sodhi YS, Steinbiss HH, Schell J, Pental D (1991) Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake. Plant Cell Rep 10(8):375–379. https://doi.org/10.1007/BF00232604

    CAS  Article  PubMed  Google Scholar 

  24. Ono Y, Takahata Y, Kaizuma N (1994) Effect of genotype on shoot regeneration from cotyledonary explants of rapeseed (Brassica napus L.). Plant Cell Rep 14(1):13–17. https://doi.org/10.1007/BF00233290

    CAS  Article  PubMed  Google Scholar 

  25. Poulsen GB (1996) Genetic transformation of Brassica. Plant Breed 115(4):209–225. https://doi.org/10.1111/j.1439-0523.1996.tb00907.x

    CAS  Article  Google Scholar 

  26. Radchuk VV, Ryschka U, Schumann G, Klocke E (2002) Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts. Physiol Plant 114(3):429–438. https://doi.org/10.1034/j.1399-3054.2002.1140313.x

    CAS  Article  PubMed  Google Scholar 

  27. Rahman H, Bennett RA, Kebede B (2017) Mapping of days to flower and seed yield in spring oilseed Brassica napus carrying genome content introgressed from Brassica oleracea. Mol Breed 37(1). https://doi.org/10.1007/s11032-016-0608-2

  28. Raineri DM, Bottino P, Gordon MP, Nester EW (1990) Agrobacterium–mediated transformation of rice (Oryza sativa L.). Nat Biotechnol 8(1):33–38. https://doi.org/10.1038/nbt0190-33

    CAS  Article  Google Scholar 

  29. Rani T, Yadav R, Yadav N, Rani A, Singh D (2013) Genetic transformation in oilseed brassicas -a review. Indian J Agric Sci 83(4):367–373

    CAS  Google Scholar 

  30. Sitther V, Tabatabai B, Enitan O, Dhekney S (2018) Agrobacterium-mediated transformation of Camelina sativa for production of transgenic plants. J Biol Methods 5(1):e83. https://doi.org/10.14440/jbm.2018.208

    Article  PubMed  PubMed Central  Google Scholar 

  31. Tang T, Yu X, Yang H, Gao Q, Ji H, Wang Y, Yan G, Peng Y, Luo H, Liu K, Li X, Ma C, Kang C, Dai C (2018) Development and validation of an effective CRISPR/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species. Front Plant Sci 9:1533. https://doi.org/10.3389/fpls.2018.01533

    Article  PubMed  PubMed Central  Google Scholar 

  32. Tzfira T, Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 17(2):147–154. https://doi.org/10.1016/j.copbio.2006.01.009

    CAS  Article  PubMed  Google Scholar 

  33. Verma S, Chinnusamy V, Kc B (2008) A simplified floral dip method for transformation of Brassica napus and B. carinata. J Plant Biochem Biotechnol 17:197–200. https://doi.org/10.1007/BF03263286

    CAS  Article  Google Scholar 

  34. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327. https://doi.org/10.1186/s12870-014-0327-y

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Yang H, Wu JJ, Tang T, Liu KD, Dai C (2017) CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus. Sci Rep 7(1):7489. https://doi.org/10.1038/s41598-017-07871-9

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7):1565–1572. https://doi.org/10.1038/nprot.2007.199

    CAS  Article  PubMed  Google Scholar 

  37. Yu B, Boyle K, Zhang W, Robinson SJ, Higgins E, Ehman L, Relf-Eckstein J-A, Rakow G, Parkin IAP, Sharpe AG, Fobert PR (2016) Multi-trait and multi-environment QTL analysis reveals the impact of seed colour on seed composition traits in Brassica napus. Mol Breed 36(8). https://doi.org/10.1007/s11032-016-0521-8

  38. Zhang K, He J, Liu L, Xie R, Qiu L, Li X, Yuan W, Chen K, Yin Y, Kyaw MMM, San AA, Li S, Tang X, Fu C, Li M (2020) A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus. Plant Methods 16:43. https://doi.org/10.1186/s13007-020-00585-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Zhang Y, Singh MB, Swoboda I, Bhalla PL (2005) Agrobacterium-mediated transformation and generation of male sterile lines of Australian canola. Aust J Agric Res 56(4):353. https://doi.org/10.1071/ar04175

    Article  Google Scholar 

Download references

Acknowledgments

We are grateful to Prof. Qijun Chen at China Agricultural University for kindly providing pKSE401 and pCBC-DT1T2 vectors for constructing CRISPR/Cas9 system.

Funding

This study was supported by the National Program of Transgenic Variety Development of China (2018ZX08020-001), the National Natural Science Foundation of China (31701458), the Fundamental Research Funds for the Central Universities (2662018QD064), and the China Postdoctoral Science Foundation (2015M580651, 2016T90704).

Author information

Affiliations

Authors

Contributions

SP.L., C.D., and L.G. designed the research. YQ. L, L.L, ZL.D., SL. L., X.T., SJ. L., B.Y., W.Y., and J.W. performed the experiments. SP.L., and C.D. analyzed the data. C.D. and SP.L. wrote the manuscript. L.G. revised the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Liang Guo or Shaoping Lu.

Ethics declarations

Competing interests

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

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

Electronic supplementary material

Table S1

(DOCX 27 kb)

Figure S1

The structure diagram of vectors. (A) The structure of pCAMBIA1300S. (B) The structure of pCAMBIA1300. (C) The structure of pCAMBIA1300S-1. (JPG 2017 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dai, C., Li, Y., Li, L. et al. An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus. Mol Breeding 40, 96 (2020). https://doi.org/10.1007/s11032-020-01174-0

Download citation

Keywords

  • Rapeseed
  • Transformation method
  • Hypocotyl
  • Agrobacterium
  • Transformation efficiency