Skip to main content
SpringerLink
Log in

Factors affecting Agrobacterium tumefaciens-mediated genetic transformation of Lycium barbarum L.

  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Summary

Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation, including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included preculture of leaf explants from 3-wk-old seedlings for 3 d on the medium for callus induction followed by inoculation with Agrobacterium strain EHA101 (pIG121 Hm), co-cultivation for 3d at 24°C, and transfer to the selection regeneration medium with 50 mg l−1 kanamycin (Kan). Using this protocol, 65% L. barbarum explants gave rise to Kan-resistant and GUS-positive calli. In addition, the expression of introduced transgene (npt II) in clonal progeny was verified by formation of calli and somatic embryos from leaf segments of nine transgenic plants grown on the Kan-containing medium. All explants formed calli at 50 mg l−1 Kan and seven out of nine transgenic plants were found to possess callus-forming capacity even at 100 mg l−1 Kan. These calli also possessed higher SE potential on SE medium supplemented with 25 mg l−1 Kan.

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

Similar content being viewed by others

References

  • An, G. High-efficiency transformation of cultured tobacco cells. Plant Physiol. 79:568–570; 1985.

    Article  PubMed  Google Scholar 

  • Arencibia, A. D.; Carmona, E. R.; Cornidel, M. T.; Castiglione, S.; O'Relly, J.; Chinea, A.; Oramas, P.; Sala, F. Somaclonal variation in insect-resistant transgenic sugarcane (Saccharum hybrid) plants produced by cell electroporation. Transgenic Res. 8:349–360; 1999.

    Article  CAS  Google Scholar 

  • Bond, J. E.; Roose, M. L. Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange. Plant Cell Rep. 18:229–234; 1998.

    Article  CAS  Google Scholar 

  • Bonneau, L.; Beranger-Novat, N.; Monin, J. Somatic embryogenesis and plant regeneration in a woody species: the European spindle tree (Euonymus europeaus L.). Plant Cell Rep. 13:135–138; 1994.

    Article  CAS  Google Scholar 

  • Cao, X.; Liu, Q.; Rowland, L. J.; Hammerschlag, F. A. GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency. Plant Cell Rep. 18:266–270; 1998.

    Article  CAS  Google Scholar 

  • Cervera, M.; Pina, J. A.; Juarez, J.; Navarro, L.; Pena, L. Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep. 18:271–278; 1998.

    Article  CAS  Google Scholar 

  • Committee of Chinese Pharmacopoeia. Chinese pharmacopoeia (I). Beijing: Chemical Industry Press; 2005: 82, 174.

    Google Scholar 

  • Curtis, I. S.; Power, J. B.; Hedden, P.; Ward, D. A.; Phillips, A.; Lower, K. C.; Davey, M. R. A stable transformation system for the ornamental plant, Datura meteloides (D.C.). Plant Cell Rep. 18:554–560; 1999.

    Article  CAS  Google Scholar 

  • Donaldson, P. A.; Simmonds, D. H. Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean. Plant Cell Rep. 19:478–484; 2000.

    Article  CAS  Google Scholar 

  • Du, L. Q.; Wang, H. Z.; Huang, F. C.; Li, A. S.; Shou, Q. Q. Genetic transformation of Lycium barbarum L. via Agrobacterium tumefaciens. Sci. China (Ser. B) 37:286–293; 1994.

    CAS  Google Scholar 

  • Fullner, K. J.; Lara, J. C.; Nester, E. W. Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273:1107–1109; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Giri, C. C.; Shyamkumar, B.; Anjaneyulu, C. Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview. Trees 18:115–135; 2004.

    Google Scholar 

  • Godwin, I.; Todd, G.; Ford-Lloyd, B.; Newbury, H. J. The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep. 9:671–675; 1991.

    Article  CAS  Google Scholar 

  • Guo, G. Q.; Frank, M.; Petra, L.; Hans-Henning, S. Factors influencing T-DNA transfer into wheat and barley cells by Agrobacterium tumefaciens. Cereal Res. Comm. 26:15–22; 1998.

    Google Scholar 

  • Hobbs, S. A. L.; Warkentin, T. M.; DeLong, C. M. O. Transgene copynumber can be positively or negatively associated with transgene expression. Plant Mol. Biol. 21:17–26; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Hoekema, A.; Hirsch, P. R.; Hooykaas, P. J.; Schilperoot, R. A. A binary plant vector strategy based on separation of vir and T-region of the A. tumefaciens Ti plasmid. Nature 303:179–180; 1983.

    Article  CAS  Google Scholar 

  • Hood, E. E.; Helmer, G. L.; Freley, R. T.; Chilton, M. D. The hyperviruence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168:1291–1301; 1986.

    PubMed  CAS  Google Scholar 

  • Hu, Z.; Guo, G. Q.; Zhao, D. L.; Li, L. H.; Zheng, G. C. Shoot regeneration from leaf explant of Lycium barbarum and Agrobacterium-mediated genetic transformation. Russ. J. Plant Physiol. 48:453–458; 2001.

    Article  CAS  Google Scholar 

  • Hu, Z.; Yang, J.; Guo, G. Q.; Zheng, G. C. High efficiency transformation of Lycium barbarum mediated by Agrobacterium tumefaciens and transgenic plant regeneration via somatic embryogenesis. Plant Cell Rep. 21:233–237; 2002.

    Article  CAS  Google Scholar 

  • Institute of Material Medica; Chinese Academy of Medical Science and Company of Chinese Medical Materials in ShanDong Province (PR China) Preliminary study on black berry of Lycium barbarum. Plant Protection 6:24–26; 1980. (in Chinese).

    Google Scholar 

  • Jain, S. M.; Gupta, P. K.; Newton, R. J. Somatic embryogenesis in woody plants, vol. 6. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2000.

    Google Scholar 

  • James, D. J.; Uratsu, S.; Cheng, J.; Negri, P.; Viss, P.; Dandekar, A. M. Acetosyringone and osmoprotectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple. Plant Cell Rep. 12:559–563; 1993.

    Article  CAS  Google Scholar 

  • Jefferson, R. A.; Kavanagh, T. A.; Bevan, M. W. GUS fusion: β-glucuronidase is a sensitive and versatile fusion marker in higher plants. EMBO J. 6:3901–3907; 1987.

    PubMed  CAS  Google Scholar 

  • Jørgensen, R. Altered gene expression in plants due to trans interaction between homologous genes. Trends Biotechnol. 8:340–344; 1990.

    Article  Google Scholar 

  • Kaneyoshi, J.; Kobayashi, S.; Nakamura, Y.; Shigemoto, N.; Doi, Y. A simple and efficient gene transfer system of trifoliate organe (Poncirus trifoliata L. Raf.). Plant Cell Rep. 13:541–545; 1994.

    Article  CAS  Google Scholar 

  • Koncz, C.; Schell, J. The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204:383–387; 1986.

    Article  CAS  Google Scholar 

  • Kudirka, D. T.; Colburn, M. A.; Hinchee, M. A.; Wright, M. S. Interactions of Agrobacterium tumefaciens with soybean (Glycine max (L.) Merrill) leaf explants in tissue culture. Can. J. Genet. Cytol. 28:808–817; 1986.

    Google Scholar 

  • Manickavasagam, M.; Ganapathi, A.; Anbazhagan, V. R.; Sudhakar, B.; Selvaraj, N.; Vasudevan, A.; Kasthurirengan, S. Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Rep. 23:134–143; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Mannerlöf, M.; Tenning, P. Variability of gene expression in transgenic tobacco. Euphytica 98:133–139; 1997.

    Article  Google Scholar 

  • McHughen, A.; Jordan, M.; Feist, G. A preculture period prior to Agrobacterium inoculation increases production of transgenic plants. J. Plant Physiol. 135:245–248; 1989.

    Google Scholar 

  • Moore, G. A.; Jacono, C. C.; Neidigh, J. L.; Lawrence, S. D.; Cline, K. Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants. Plant Cell Rep. 11:238–242; 1992.

    Article  CAS  Google Scholar 

  • Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays of tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.

    Article  CAS  Google Scholar 

  • Pena, L.; Cervera, M.; Juarez, J.; Navarro, A.; Pina, J. A.; Navarro, L. Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration. Plant Cell Rep. 16:731–737; 1997.

    Article  CAS  Google Scholar 

  • Rashid, H.; Yokoi, S.; Toriyama, K.; Hinata, K. Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep. 15:727–730; 1996.

    Article  CAS  Google Scholar 

  • Rogers, H. J.; Parkers, H. C. Transgenic plants and the environment. J. Exp. Bot. 46:467–488; 1995.

    Article  CAS  Google Scholar 

  • Salas, M. G.; Park, S. H.; Srivatanakul, M.; Smith, R. H. Temperature influence on stable T-DNA integration in plant cells. Plant Cell Rep. 20:701–705; 2001.

    Article  CAS  Google Scholar 

  • Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning—a laboratory manual, 2nd edn. New York: Cold Spring Harbor Laboratory Press; 1989.

    Google Scholar 

  • Sangwan, R. S.; Bourgeois, Y.; Brown, S.; Vasseur, G.; Sangwan-Noreel, B. S. Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana. Planta 188:439–456; 1992.

    Article  CAS  Google Scholar 

  • Sangwan, R. S.; Bourgeois, Y.; Sangwan-Norreel, B. S. Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency. Mol. Gen. Genet. 230:475–485; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Sriskandarajah, S.; Goodwin, P. Conditioning promotes regeneration and transformation in apple leaf explants. Plant Cell Tiss. Organ Cult. 53:1–11; 1998.

    Article  CAS  Google Scholar 

  • Tohidfar, M.; Mohammadi, M.; Ghareyazie, B. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) using a heterologous bean chitinase gene. Plant Cell Tiss. Organ Cult. 83:83–96; 2005.

    Article  CAS  Google Scholar 

  • Uranbey, S.; Sevýmay, C. S.; Kaya, M. D.; Ýpek, A.; Sancak, C.; Ba§alma, D.; Er, C.; Özcan, S. Influence of different co-cultivation temperatures, periods and media on Agrobacterium tumefaciens-mediated gene transfer. Biol. Plant. 49:53–57; 2005.

    Article  Google Scholar 

  • Vancanneyt, G.; Schidt, R.; O'Connor-Sanchez, A.; Willmitzer, L.; Rocha-Sosa, M. Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220:245–250; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Villemont, E.; Dubois, F.; Sangwan, R. S.; Vasseur, G.; Bourgeois, Y.; Sangwan-Noreel, B. S. Role of the host cell cycle in the Agrobacterium-mediated transformation of Petunia: evidence of an S-phase control mechanism for T-DNA transfer. Planta 201:160–172; 1997.

    Article  CAS  Google Scholar 

  • Xie, D. Y.; Hong, Y. Agrobacterium-mediated genetic transformation of Acacia mangium. Plant Cell Rep. 20:917–922; 2002.

    Article  CAS  Google Scholar 

  • Yu, M. S.; Yuen, W. H.; So, K. F.; Chang, R. C. C. Neuroprotective effects of extracts from anti-aging Chinese medicine Lycium barbarum (Gou-Qi-Zi). J. Neurochem. 88(Suppl.): 69; 2004.

    Google Scholar 

  • Zhang, M.; Chen, H. X.; Huang, J.; Li, Z.; Zhu, C. P.; Zhang, S. H. Effect of Lycium barbarum polysaccharide on human hepatoma QGY7703 cells: inhibition of proliferation and induction of apoptosis. Life Sci. 76:2115–2124; 2005.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Shantou University, 515063, Shantou, People's Republic of China

    Zhong Hu & Yi-Rui Wu

  2. Institute of Cell Biology, Lanzhou University, 730000, Lanzhou, People's Republic of China

    Wei Li & Huan-Huan Gao

Authors
  1. Zhong Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi-Rui Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Huan-Huan Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhong Hu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hu, Z., Wu, YR., Li, W. et al. Factors affecting Agrobacterium tumefaciens-mediated genetic transformation of Lycium barbarum L.. In Vitro Cell.Dev.Biol.-Plant 42, 461–466 (2006). https://doi.org/10.1079/IVP2006796

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1079/IVP2006796

Key words

Search

Navigation