Article

Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers

Received:
Accepted:
Published online:

Abstract

Genetic modification plays a vital role in breeding new crops with excellent traits. Almost all the current genetic modification methods require regeneration from tissue culture, involving complicated, long and laborious processes. In particular, many crop species such as cotton are difficult to regenerate. Here, we report a novel transformation platform technology, pollen magnetofection, to directly produce transgenic seeds without regeneration. In this system, exogenous DNA loaded with magnetic nanoparticles was delivered into pollen in the presence of a magnetic field. Through pollination with magnetofected pollen, transgenic plants were successfully generated from transformed seeds. Exogenous DNA was successfully integrated into the genome, effectively expressed and stably inherited in the offspring. Our system is culture-free and genotype independent. In addition, it is simple, fast and capable of multi-gene transformation. We envision that pollen magnetofection can transform almost all crops, greatly facilitating breeding processes of new varieties of transgenic crops.

  • Subscribe to Nature Plants for full access:

    $62

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Ahmad, P. et al. Role of transgenic plants in agriculture and biopharming. Biotechnol. Adv. 30, 524–540 (2012).

  2. 2.

    Ashraf, M. & Akram, N. A. Improving salinity tolerance of plants through conventional breeding and genetic engineering: An analytical comparison. Biotechnol. Adv. 27, 744–752 (2009).

  3. 3.

    Rojas, C. A., Hemerly, A. S. & Ferreira, P. C. Genetically modified crops for biomass increase. Genes and strategies. GM Crops 1, 137–142 (2010).

  4. 4.

    Newell, C. A. Plant transformation technology. Developments and applications. Mol. Biotechnol. 16, 53–65 (2000).

  5. 5.

    Rivera, A. L., Gómez-Lim, M., Fernández, F. & Loske, A. M. Physical methods for genetic plant transformation. Phys. Life Rev. 9, 308–345 (2012).

  6. 6.

    Rao, A. Q. et al. The myth of plant transformation. Biotechnol. Adv. 27, 753–763 (2009).

  7. 7.

    Taylor, N. J. & Fauquet, C. M. Microparticle bombardment as a tool in plant science and agricultural biotechnology. DNA Cell Biol. 21, 963–977 (2002).

  8. 8.

    Hansen, G. & Wright, M. S. Recent advances in the transformation of plants. Trends Plant Sci. 4, 226–231 (1999).

  9. 9.

    Juturu, V. N., Mekala, G. K. & Kirti, P. B. Current status of tissue culture and genetic transformation research in cotton (Gossypium spp.). Plant Cell Tiss. Organ. Cult. 120, 813–839 (2014).

  10. 10.

    Trolinder, N. L. & Goodin, J. R. Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Rep. 6, 231–234 (1987).

  11. 11.

    Trolinder, N. L. & Xhixian, C. Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Rep. 8, 133–136 (1989).

  12. 12.

    Christou, P. Transformation technology. Trends Plant Sci. 1, 423–431 (1996).

  13. 13.

    Morre, J. L., Permingeat, H. R., Romagnoli, M. V., Heisterborg, C. M. & Vallejos, R. H. Multiple shoot induction and plant regeneration from embryonic axes of cotton. Plant Cell Tiss. Org. 54, 131–136 (1998).

  14. 14.

    Krishna, G., Reddy, P. S., Ramteke, P. W. & Bhattacharya, P. S. Progress of tissue culture and genetic transformation research in pigeon pea (Cajanus cajan (L.) Millsp.). Plant Cell Rep. 29, 1079–1095 (2010).

  15. 15.

    Stöger, E., Moreno, R. M. B., Ylstra, B., Vicente, O. & Heberle-Bors, E. Comparison of different techniques for gene transfer into mature and immature tobacco pollen. Transgen. Res. 1, 71–78 (1992).

  16. 16.

    Schreiber, D. N. & Dresselhaus, T. In vitro pollen germination and transient transformation of Zea mays and other plant species. Plant Mol. Biol. Rep. 21, 31–41 (2003).

  17. 17.

    Wang, W. Q. et al. Pollen-mediated transformation of Sorghum bicolor plants. Biotechnol. Appl. Bioc. 48, 79–83 (2007).

  18. 18.

    Ohta, Y. High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA. Proc. Natl Acad. Sci.  USA  83, 715–719 (1986).

  19. 19.

    Touraev, A., Stöger, E., Voronin, V. & Heberle-Bors, E. Plant male germ line transformation. Plant J. 12, 949–956 (1997).

  20. 20.

    Abdul-Baki, A. A., Saunders, J. A., Matthews, B. F. & Pittarelli, G. W. DNA uptake during electroporation of germinating pollen grains. Plant Sci. 70, 181–190 (1990).

  21. 21.

    Aronen, T. S., Nikkanen, T. O. & Häggman, H. M. Compatibility of different pollination techniques with microprojectile bombardment of Norway spruce and Scots pine pollen. Can. J. For. Res. 28, 79–86 (1998).

  22. 22.

    Barinova, L. et al. Antirrhinum majus microspore maturation and transient transformation in vitro. J. Exp. Bot. 53, 1119–1129 (2002).

  23. 23.

    Fernando, D. D., Owens, J. N. & Misra, S. Transient gene expression in pine pollen tubes following particle bombardment. Plant Cell Rep. 19, 224–228 (2000).

  24. 24.

    Folling, L. & Olesen, A. Transformation of wheat (Triticum aestivum L.) microspore-derived callus and microspores by particle bombardment. Plant Cell Rep. 23, 629–636 (2001).

  25. 25.

    Tjokrokusumo, D., Heinrich, T., Wylie, S., Potter, R. & McComb, J. Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation. Plant Cell Rep. 19, 792–797 (2000).

  26. 26.

    Kumlehn, J., Serazetdinova, L., Hensel, G., Becker, D. & Loerz, H. Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotechnol. J. 4, 251–261 (2006).

  27. 27.

    Kim, S.-S., Shin, D.-I. & Park, H.-S. Transient β-glucuronidase expression in lily (Lilium longflorum L.) pollen via wounding-assisted Agrobacterium-mediated transformation. Biotechnol. Lett. 29, 965–969 (2007).

  28. 28.

    Wang, H. & Jiang, L. W. Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes. Nat. Protoc. 6, 419–426 (2011).

  29. 29.

    Ali, A., Bang, S. W., Chung, S. M. & Staub, J. E. Plant transformation via pollen tube-mediated gene transfer. Plant Mol. Bio. Rep. 33, 742–747 (2015).

  30. 30.

    Eapen, S. Pollen grains as a target for introduction of foreign genes into plants: an assessment. Physiol. Mol. Biol. Plants 17, 1–8 (2011).

  31. 31.

    Torney, F., Trewyn, B. G., Lin, V. S. & Wang, K. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat. Nanotechnol. 2, 295–300 (2007).

  32. 32.

    Scherer, F. et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther. 9, 102–109 (2002).

  33. 33.

    Dobson, J. Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery. Gene Ther. 13, 283–287 (2006).

  34. 34.

    Plank, C., Zelphati, O. & Mykhaylyk, O. Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects. Adv. Drug Deliv. Rev. 63, 1300–1331 (2011).

  35. 35.

    Hao, R. et al. Synthesis, functionalization, and biomedical applications of multifunctional magnetic nanoparticles. Adv. Mater. 22, 2729–2742 (2010).

  36. 36.

    Tseng, P., Judy, J. W. & Di Carlo, D. Magnetic nanoparticle-mediated massively parallel mechanical modulation of single-cell behavior. Nat. Methods 9, 1113–1119 (2012).

  37. 37.

    Wang, Y. et al. A magnetic nanoparticle-based multiple-gene delivery system for transfection of porcine kidney cells. Plos ONE 9, e102886 (2014).

  38. 38.

    Chen, W. et al. Characterization and insights into the nano liposomal magnetic gene vector used for cell co-transfection. J. Nanosci. Nanotechnol. 15, 1–7 (2014).

  39. 39.

    Zhao, X. et al. Morphology, structure and function characterization of PEI modified magnetic nanoparticles gene delivery system. Plos ONE 9, e98919 (2014).

  40. 40.

    Wang, Y. et al. Study on performance of magnetic fluorescent nanoparticles as gene carrier and location in pig kidney cells. Nanoscale Res. Lett. 8, 127 (2013).

  41. 41.

    Ressayre, A., Godelle, B., Mignot, A. & Gouyon, Ph A morphogenetic model accounting for pollen aperture pattern in flowering plants. J. Theor. Biol. 193, 321–334 (1998).

  42. 42.

    Kakani, V. G. et al. Differences in in vitro pollen germination and pollen tube growth of cotton cultivars in response to high temperature. Ann. Bot. 96, 59–67 (2005).

  43. 43.

    Wang, Y., Zhang, R., Zu, M. T. & Guo, S. D. Bt-Cpti insect-resistant hybrid cotton: YingMian 2. China Cotton 9, 20 (2005).

  44. 44.

    Liu, Z. L., Jiang, W. C., Su, J. Q. & Zhang, H. Y. A new variety of disease-resistant and high yield cotton: SU12. Jiangsu Agr. Sci. 4, 25–26 (1997).

  45. 45.

    Singh, M. & Bhalla, P. Control of male germ-cell development in flowering plants. Bioessays 29, 1124 (2007).

  46. 46.

    R Core Team R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria, 2014); https://www.r-project.org/

Download references

Acknowledgements

This research was supported by the Major National Scientific Research Program of China (2014CB932200), the Genetically Modified Organisms Breeding Major Projects of China (No. 2009ZX08010-006B), the Agricultural Science and Technology Innovation Program (CAASXTCX2016004), the National Natural Science Foundation of China (No. 31301373), the Beijing Municipal Natural Science Foundation (6164045) and the Genetically Modified Organisms Breeding Major Projects of China (No. 2011ZX08005-004).

We thank Q. Wu and C. X. Wang of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences for pepper pollen, pumpkin pollen and cocozelle pollen.

Author information

Author notes

  1. Xiang Zhao, Zhigang Meng and Yan Wang contributed equally to this work.

Affiliations

  1. Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China

    • Xiang Zhao
    • , Yan Wang
    • , Wenjie Chen
    • , Changjiao Sun
    • , Bo Cui
    • , Jinhui Cui
    • , Manli Yu
    • , Zhanghua Zeng
    •  & Haixin Cui
  2. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China

    • Zhigang Meng
    • , Sandui Guo
    •  & Rui Zhang
  3. Nanobiotechnology Research Center, Chinese Academy of Agricultural Sciences, Beijing, China

    • Xiang Zhao
    • , Zhigang Meng
    • , Yan Wang
    • , Wenjie Chen
    • , Changjiao Sun
    • , Bo Cui
    • , Jinhui Cui
    • , Manli Yu
    • , Zhanghua Zeng
    •  & Haixin Cui
  4. Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA

    • Dan Luo
  5. Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, USA

    • Dan Luo
  6. Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, China

    • Dan Luo
  7. Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA

    • Jerry Q. Cheng

Authors

  1. Search for Xiang Zhao in:

  2. Search for Zhigang Meng in:

  3. Search for Yan Wang in:

  4. Search for Wenjie Chen in:

  5. Search for Changjiao Sun in:

  6. Search for Bo Cui in:

  7. Search for Jinhui Cui in:

  8. Search for Manli Yu in:

  9. Search for Zhanghua Zeng in:

  10. Search for Sandui Guo in:

  11. Search for Dan Luo in:

  12. Search for Jerry Q. Cheng in:

  13. Search for Rui Zhang in:

  14. Search for Haixin Cui in:

Contributions

H.C., S.G., R.Z. and D.L. conceived the experiment. X.Z. performed pollen transformation system construction and tracking of MNP–DNA complexes in pollen; Z.M. performed vector construction and promoter analysis; X.Z., Z.M., Y.W.,W.C. and M.Y. performed pollen transformation and transgenic plant analysis; Z.M., X.Z., W.C., C.S. and J.C. performed the field trial; X.Z., Y.W., B.C. and Z.Z. analysed the data; H.C., D.L., X.Z. and Y.W. wrote the paper; J.Q.C. performed the statistical analyses.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rui Zhang or Haixin Cui.

Electronic supplementary material

  1. Supplementary Information

    Supplementary Figures 1–37, Supplementary Tables 1–6, Supplementary References

  2. Life Sciences Reporting Summary