Czech J. Genet. Plant Breed., 2020, 56(1):34-41 | DOI: 10.17221/153/2018-CJGPB

The application of an Agrobacterium-mediated in planta transformation system in a Catharanthus roseus medicinal plantOriginal Paper

Zahra Bahari1, Sima Sazegari2, Ali Niazi*,1, Alireza Afsharifar3
1 Institute of Biotechnology, Shiraz University, Shiraz, Iran
2 Department of Crop Biotechnology and Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
3 Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran

The lack of an efficient protocol for the transformation of Catharanthus roseus, as an important medicinal plant, causes obstacles in the genetic engineering programmes of this plant. Therefore, the present study was conducted to examine a convenient and reliable system for generating stable transgenic lines based on the in planta method. To do so, two different in planta Agrobacterium infection systems were investigated. The transgenic lines were produced through ovary injection and shoot apical meristem (SAM) immersing methods. The presence and expression of the GFP transgene in the T0 and T1 lines were confirmed by PCR and RT-PCR. Moreover, the ELISA test confirmed the GFP expression in the transgenic lines. Apart from the superiority of the ovary injection over the SAM inoculation due to the normal growth and healthy appearance, it also showed a significant difference from the point of the confirmed transgenic plant numbers. GFP integration was confirmed in 6 out of 50 T1 plants from the ovary injection and only 3 out of 150 plants generated from the SAM inoculation. In addition to the comparison of these two separate methods, we reached a new convenient in planta system by a 12% transformation rate for generating the C. roseus transgenic lines. According to the result of this study, ovary injection can be introduced as a novel, facile, and stable method for the Catharanthus transformation programme.

Keywords: gene expression; GFP gene; ovary injection; shoot apical meristem infection; transformation rate

Published: March 31, 2020  Show citation

ACS AIP APA ASA Harvard Chicago Chicago Notes IEEE ISO690 MLA NLM Turabian Vancouver
Bahari Z, Sazegari S, Niazi A, Afsharifar A. The application of an Agrobacterium-mediated in planta transformation system in a Catharanthus roseus medicinal plant. Czech J. Genet. Plant Breed. 2020;56(1):34-41. doi: 10.17221/153/2018-CJGPB.
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. Alam P., Khan Z.A., Abdin M.Z., Khan J.A., Ahmad P., Elkholy S.F., Sharaf-Eldin M.A. (2017): Efficient regeneration and improved sonication-assisted Agrobacterium transformation (SAAT) method for Catharanthus roseus. 3 Biotech, 7: 26-36. Go to original source... Go to PubMed...
    2. Arthikala M.K., Nanjareddy K., Lara M., Sreevathsa R. (2014): Utility of a tissue culture-independent Agrobacterium mediated In planta transformation strategy in bell pepper to develop fungal disease resistant plants. Scientia Horticulturae, 170: 61-69. Go to original source...
    3. Ben-Amar A., Cobanov P., Buchholz G., Mliki A., Reustle G. (2013): In planta agro-infiltration system for transient gene expression in grapevine (Vitis spp.). Acta Physiologiae Plantarum, 35: 3147-3156. Go to original source...
    4. Canel C., Lopes-Cardoso M.I., Whitmer S., van der Fits L., Pasquali G., van der Heijden R., Verpoorte R. (1998): Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta, 205: 414-419. Go to original source... Go to PubMed...
    5. Choi P.S., Kim Y.D., Choi K.M., Chung H.J., Choi D.W., Liu J.R. (2004): Plant regeneration from hairy-root cultures transformed by infection with Agrobacterium rhizogenes in Catharanthus roseus. Plant Cell Reports, 22: 828-831. Go to original source... Go to PubMed...
    6. Clough S.J., Bent A.F. (1998): Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16: 735-743. Go to original source... Go to PubMed...
    7. Dhandapani M., Kim D.H., Hong S.B. (2007): Transgenic plant development in Catharanthus roseus: limiting factors and scope. Transgenic Plant Journal, 1: 163-168.
    8. Feldmann K.A., Marks M.D. (1987): Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Molecular and General Genetics, 208: 1-9. Go to original source...
    9. Gawel N., Jarret R. (1991): A modified CTAB DNA extraction procedure for Musa and Ipomoea. Plant Molecular Biology Report, 9: 262-266. Go to original source...
    10. Guimaraes G., Cardoso L., Oliveira H Santos C., Duarte P., Sottomayor M. (2012): Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don. AoB Plants, 2012: pls002. Go to original source...
    11. Jaganath B., Subramanyam K., Mayavan S., Karthik S., Elayaraja D., Udayakumar R., Ganapathi A. (2014): An efficient In planta transformation of Jatropha curcas (L.) and multiplication of transformed plants through in vivo grafting. Protoplasma, 251:591-601. Go to original source... Go to PubMed...
    12. Kalbande B.B., Patil A.S. (2016): Plant tissue culture independent Agrobacterium tumefaciens mediated in planta transformation strategy for upland cotton (Gossypium hirsutum). Journal of Genetic Engineering and Biotechnology, 14: 9-18. Go to original source... Go to PubMed...
    13. Keshamma E., Rohini S., Rao K., Madhusudhan B., Kumar M.U. (2008): Tissue culture-independent in planta transformation strategy: an Agrobacterium tumefaciensmediated gene transfer method to overcome recalcitrance in cotton (Gossypium hirsutum L.). Journal of Cotton Science, 12: 264-272.
    14. Kruger N.J. (2002): The Bradford method for protein quantitation. In: Walker J.M. (ed.): The Protein Protocols Handbook. Totowa, Humana Press: 17-24. Go to original source...
    15. Liu M., Yang J., Cheng Y.Q., An L.J. (2009): Optimization of soybean (Glycine max (L.) Merrill) in planta ovary transformation using a linear minimal gus gene cassette. Journal of Zhejiang University Science, 10: 870-876. Go to original source... Go to PubMed...
    16. Magnotta M., Murata J., Chen J., De Luca V. (2006): Identification of a low vindoline accumulating cultivar of Catharanthus roseus (L.) G. Don by alkaloid and enzymatic profiling. Phytochemistry, 67: 1758-1764. Go to original source... Go to PubMed...
    17. Makhzoum A., Bjelica A., Petit-Paly G., Bernards M.A. (2015): Novel plant regeneration and transient gene expression in Catharanthus roseus. Biology, 6: 1-9.
    18. Muzamli A., Mujib A., Tonk D., Zafar N. (2016): Plant regeneration through somatic embryogenesis and genome size analysis of Coriandrum sativum L. Protoplasma, 254: 1-10. Go to original source... Go to PubMed...
    19. Shah S.H., Ali S., Jan S.A., Ali G.M. (2015): Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.). Plant Cell, Tissue and Organ Culture, 120: 1139-1157. Go to original source...
    20. Supartana P., Shimizu T., Nogawa M., Shioiri H., Nakajima T., Haramoto N., Kojima M. (2006): Development of simple and efficient in planta transformation method for wheat (Triticum aestivum L.) using Agrobacterium tumefaciens. Journal of Bioscience and Bioengineering, 102: 162-170. Go to original source... Go to PubMed...
    21. Tjokrokusumo D., Heinrich T., Wylie S., Potter R., McComb J. (2000): Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation. Plant Cell Reports, 19: 792-797. Go to original source... Go to PubMed...
    22. Verma P., Mathur A.K. (2011): Agrobacterium tumefaciens-mediated transgenic plant production via direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus roseus. Biotechnology Letters, 33: 1053-1060. Go to original source... Go to PubMed...
    23. Wang S., Guo A., Zheng W., Zhang Y., Qiao H., Kennedy I. (2007): Development of ELISA for the determination of transgenic Bt-cottons using antibodies against Cry1Ac protein from Bacillus thuringiensis HD-73. Engineering in Life Sciences, 7: 149-154. Go to original source...
    24. Wang Q., Xing S., Pan Q., Yuan F., Zhao J., Tian Y., Tang K. (2012): Development of efficient Catharanthus roseus regeneration and transformation system using Agrobacterium tumefaciens and hypocotyls as explants. BMC Biotechnology, 12: 34-46. Go to original source... Go to PubMed...
    25. Yasmeen A., Mirza B., Inayatullah S., Safdar N., Jamil M., Ali S., Choudhry M. F. (2009): In planta transformation of tomato. Plant Molecular Biology Reporter, 27: 20-28. Go to original source...
    26. Yellisetty V., Reddy L., Mandapaka M. (2015): In planta transformation of sorghum (Sorghum bicolor (L.) Moench) using TPS1 gene for enhancing tolerance to abiotic stresses. Journal of Genetic, 94: 425-434. Go to original source... Go to PubMed...
    27. Zhao L., Sander G.W., Shanks J.V. (2013): Perspectives of the metabolic engineering of terpenoid indole alkaloids in Catharanthus roseus hairy roots. Biotechnology of Hairy Root Systems, 134: 23-54. Go to original source... Go to PubMed...

    This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY NC 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.


    Return to the content