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.
Similar content being viewed by others
References
An, G. High-efficiency transformation of cultured tobacco cells. Plant Physiol. 79:568–570; 1985.
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.
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.
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.
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.
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.
Committee of Chinese Pharmacopoeia. Chinese pharmacopoeia (I). Beijing: Chemical Industry Press; 2005: 82, 174.
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.
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.
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.
Fullner, K. J.; Lara, J. C.; Nester, E. W. Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273:1107–1109; 1996.
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.
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.
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.
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.
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.
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.
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.
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.
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).
Jain, S. M.; Gupta, P. K.; Newton, R. J. Somatic embryogenesis in woody plants, vol. 6. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2000.
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.
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.
Jørgensen, R. Altered gene expression in plants due to trans interaction between homologous genes. Trends Biotechnol. 8:340–344; 1990.
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.
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.
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.
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.
Mannerlöf, M.; Tenning, P. Variability of gene expression in transgenic tobacco. Euphytica 98:133–139; 1997.
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.
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.
Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays of tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.
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.
Rashid, H.; Yokoi, S.; Toriyama, K.; Hinata, K. Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep. 15:727–730; 1996.
Rogers, H. J.; Parkers, H. C. Transgenic plants and the environment. J. Exp. Bot. 46:467–488; 1995.
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.
Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning—a laboratory manual, 2nd edn. New York: Cold Spring Harbor Laboratory Press; 1989.
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.
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.
Sriskandarajah, S.; Goodwin, P. Conditioning promotes regeneration and transformation in apple leaf explants. Plant Cell Tiss. Organ Cult. 53:1–11; 1998.
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.
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.
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.
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.
Xie, D. Y.; Hong, Y. Agrobacterium-mediated genetic transformation of Acacia mangium. Plant Cell Rep. 20:917–922; 2002.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Rights 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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1079/IVP2006796