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Production of Herbicide-Resistant Medicinal Plant Salvia miltiorrhiza Transformed with the Bar Gene

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Abstract

In this study, we successfully performed Agrobacterium-mediated genetic transformation of Salvia miltiorrhiza and produced herbicide-resistant transformants. Leaf discs of S. miltiorrhiza were infected with Agrobacterium tumefaciens EHA105 harboring pCAMBIA 3301. The pCAMBIA 3301 includes an intron-containing gus reporter and a bar selection marker. To increase stable transformation efficiency, a two-step selection was employed which consists of herbicide resistance and gus expression. Here, we put more attention to the screening step of herbicide resistance. The current study provides an efficient screening system for the transformed plant of S. miltiorrhiza harboring bar gene. To determine the most suitable phosphinothricin concentration for plant selection, non-transformed leaf discs were grown on selection media containing six different phosphinothricin concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/l). Based on the above results of non-transformed calluses, the sensitivity of phosphinothricin (0, 0.4, 0.8, 1.2, 1.6 mg/l) was tested in the screening of transgenic S. miltiorrhiza. We identified that 0.6 mg/l phosphinothricin should be suitable for selecting putatively transformed callus because non-transformed callus growth was effectively inhibited under this concentrations. When sprayed with Basta, the transgenic S. miltiorrhiza plants were tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to S. miltiorrhiza.

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Abbreviations

S. miltiorrhiza :

Salvia miltiorrhiza

A. tumefaciens :

Agrobacterium tumefaciens

MS salts:

Murashige and Skoog salts

CaMV 35S:

Cauliflower mosaic virus 35S promoter

LB:

Luria-Bertani broth

GUS:

β-Glucuronidase

References

  1. Chai, B., & Sticklen, M. B. (1998). Applications of biotechnology in turfgrass genetic improvement. Crop Science, 38, 1320–1338.

    Article  Google Scholar 

  2. Chai, M. I., Wang, B. I., Kim, J. Y., Lee, J. M., & Kim, D.-H. (2003). Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass. Journal of Zhejiang University SCIENCE A, 4, 346–351.

    Article  CAS  Google Scholar 

  3. Chai, M., Senthil, K., & Kim, D. (2004). Transgenic plants of colonial bentgrass from embryogenic callus via Agrobacterium-mediated transformation. Plant Cell, Tissue and Organ Culture, 77, 165–171.

    Article  CAS  Google Scholar 

  4. Chen, H., Yuan, J.-P., Chen, F., Zhang, Y.-L., & Song, J.-Y. (1997). Tanshinone production in Ti-transformed Salvia miltiorrhiza cell suspension cultures. Journal of Biotechnology, 58, 147–156.

    Article  CAS  Google Scholar 

  5. Choi, H. J., Chandrasekhar, T., Lee, H.-Y., & Kim, K.-M. (2007). Production of herbicide-resistant transgenic sweet potato plants through Agrobacterium tumefaciens method. Plant Cell, Tissue and Organ Culture, 91, 235–242.

    Article  CAS  Google Scholar 

  6. Dong, S., & Qu, R. (2005). High efficiency transformation of tall fescue with Agrobacterium tumefaciens. Plant Science, 168, 1453–1458.

    Article  CAS  Google Scholar 

  7. Fu, D., Tisserat, N. A., Xiao, Y., Settle, D., Muthukrishnan, S., & Liang, G. H. (2005). Overexpression of rice TLPD34 enhances dollar-spot resistance in transgenic bentgrass. Plant Science, 168, 671–680.

    Article  CAS  Google Scholar 

  8. Fu, D., Xiao, Y., Muthukrishnan, S., & Liang, G. H. (2005). In vivo performance of a dual genetic marker, manA-gfp, in transgenic bentgrass. Genome, 48, 722–730.

    Article  CAS  Google Scholar 

  9. Ge, Y., Cheng, X., Hopkins, A., & Wang, Z.-Y. (2007). Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation. Plant Cell Reports, 26, 783–789.

    Article  CAS  Google Scholar 

  10. Ge, Y., Norton, T., & Wang, Z.-Y. (2006). Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation. Plant Cell Reports, 25, 792–798.

    Article  CAS  Google Scholar 

  11. Gu, X.-C., Chen, J.-F., Xiao, Y., Di, P., Xuan, H.-J., Zhou, X., Zhang, L., & Chen, W.-S. (2012). Overexpression of allene oxide cyclase promoted tanshinone/phenolic acid production in Salvia miltiorrhiza. Plant Cell Reports, 31, 2247–2259.

    Article  CAS  Google Scholar 

  12. Guillon, S., Trémouillaux-Guiller, J., Pati, P. K., Rideau, M., & Gantet, P. (2006). Hairy root research: recent scenario and exciting prospects. Current Opinion in Plant Biology, 9, 341–346.

    Article  CAS  Google Scholar 

  13. Han, N., Chen, D., Bian, H.-W., Deng, M.-J., & Zhu, M.-Y. (2005). Production of transgenic creeping bentgrass Agrostis stolonifera var. palustris plants by Agrobacterium tumefaciens-mediated transformation using hygromycin selection. Plant Cell, Tissue and Organ Culture, 81, 131–138.

    Article  Google Scholar 

  14. Ho, H.-S., Vishwakarma, R. K., Chen, E. C.-F., & Tsay, H.-S. (2013). Activation tagging in Salvia miltiorrhiza can cause increased leaf size and accumulation of tanshinone I and IIA in its roots. Botanical Studies, 54, 37.

    Article  Google Scholar 

  15. Horsch, R., Fry, J., Hoffmann, N., Eichholtz, D., Rogers, S. A., & Fraley, R. (1985). A simple and general method for transferring genes into plants. Science, 227, 1229–1231.

    Article  CAS  Google Scholar 

  16. Huang, L., Dai, Z., Lv, D. and Yuan, Y. (2009) [Discuss on model organism and model for geoherbs’ study]. Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi= China Journal of Chinese Materia Medica, 34, 1063–1066.

  17. Huang, L., Liu, D., & Hu, Z. (2000). [Effects of phytohormones on growth and content of depsides in Salvia miltiorrhiza suspension cells]. Zhong yao cai= Zhongyaocai=Journal of Chinese Medicinal materials, 23, 1–4.

    CAS  Google Scholar 

  18. Kim, S. J., Lee, J.-Y., Kim, Y.-M., Yang, S.-S., Hwang, O.-J., Hong, N.-J., Kim, K.-M., Lee, H.-Y., Song, P.-S., & Kim, J.-I. (2007). Agrobacterium-mediated high-efficiency transformation of creeping bentgrass with herbicide resistance. Journal of Plant Biology, 50, 577–585.

    Article  CAS  Google Scholar 

  19. Lee, C.-Y., Agrawal, D. C., Wang, C.-S., Yu, S.-M., Chen, J. J., & Tsay, H.-S. (2008). T-DNA activation tagging as a tool to isolate Salvia miltiorrhiza transgenic lines for higher yields of tanshinones. Planta Medica, 74, 780.

    Article  CAS  Google Scholar 

  20. Lee, L. (1996). Turfgrass biotechnology. Plant Science, 115, 1–8.

    Article  Google Scholar 

  21. Lohar, D. P., Schuller, K., Buzas, D. M., Gresshoff, P. M., & Stiller, J. (2001). Transformation of Lotus japonicus using the herbicide resistance bar gene as a selectable marker. Journal of Experimental Botany, 52, 1697–1702.

    Article  CAS  Google Scholar 

  22. Lutz, K. A., Knapp, J. E., & Maliga, P. (2001). Expression of bar in the plastid genome confers herbicide resistance. Plant Physiology, 125, 1585–1590.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Ono, N. N., & Tian, L. (2011). The multiplicity of hairy root cultures: prolific possibilities. Plant Science, 180, 439–446.

    Article  CAS  Google Scholar 

  25. Sarria, R., Torres, E., Angel, F., Chavarriaga, P., & Roca, W. M. (2000). Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation. Plant Cell Reports, 19, 339–344.

    Article  CAS  Google Scholar 

  26. Thompson, C. J., Movva, N. R., Tizard, R., Crameri, R., Davies, J. E., Lauwereys, M., & Botterman, J. (1987). Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. The EMBO Journal, 6, 2519.

    CAS  Google Scholar 

  27. Wang, K., Luo, Q., & Cheng, H. (1998). [Production of allochtonic formations in callus cells of Salvia miltiorrhiza Bge.]. Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi=China Journal of Chinese Materia Medica, 23(592–594), 638.

    Google Scholar 

  28. Wang, Z.-Y., & Ge, Y. (2005). Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea). Journal of Plant Physiology, 162, 103–113.

    Article  CAS  Google Scholar 

  29. Wang, Z.-Y., & Ge, Y. (2006). Recent advances in genetic transformation of forage and turf grasses. In Vitro Cellular & Developmental Biology-Plant, 42, 1–18.

    Article  Google Scholar 

  30. Wang, Z., Hopkins, A., & Mian, R. (2001). Forage and turf grass biotechnology. Critical Reviews in Plant Sciences, 20, 573–619.

    Article  CAS  Google Scholar 

  31. Wu, J.-Y., & Shi, M. (2008). Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of Salvia miltiorrhiza hairy roots. Applied Microbiology and Biotechnology, 78, 441–448.

    Article  CAS  Google Scholar 

  32. Yan, Y. P., & Wang, Z. Z. (2007). Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method. Plant Cell, Tissue and Organ Culture, 88, 175–184.

    Article  CAS  Google Scholar 

  33. Yu, T., Skinner, D., Liang, G., Trick, H., Huang, B., & Muthukrishnan, S. (2001). Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene. Hereditas, 133, 229–223.

    Article  CAS  Google Scholar 

  34. Yuan, J., Tao, L., & Xu, J. (1989). Immobilization of callus tissue cells of Salvia miltiorrhiza and the characteristics of their products. Chinese Journal of Biotechnology, 6, 199–205.

    Google Scholar 

  35. Zhang, Y., Song, J., Qi, J., & Lu, G. (1997). [The plant regeneration of Salvia miltiorrhiza Bge. transformed by Agrobacterium]. Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi=China Journal of Chinese Materia Medica, 22(274–275), 318.

    CAS  Google Scholar 

  36. Zhang, Y., Song, J., Zhao, B., & Liu, H. (1994). Crown gall culture and production of tanshinone in Salvia miltiorrhiza. Chinese Journal of Biotechnology, 11, 137–141.

    Google Scholar 

  37. Zhang, Y., Yan, Y.-P., Wu, Y.-C., Hua, W.-P., Chen, C., Ge, Q., & Wang, Z.-Z. (2014). Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation. Metabolic Engineering, 21, 71–80.

    Article  Google Scholar 

  38. Zhong, H., Bolyard, M. G., Srinivasan, C., & Sticklen, M. B. (1993). Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic callus. Plant Cell Reports, 13, 1–6.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the Sichuan Provincial Science and Technology Department (no. 2011NZ0098-12-02) to DKJ and ZXL.

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Conflicts of Interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Chengdu, Sichuan, China

    Yu Liu, Shi Xin Yang, Yan Cheng, Dong Qing Liu, Yong Zhang, Ke Jun Deng & Xue Lian Zheng

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  1. Yu Liu
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  2. Shi Xin Yang
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  3. Yan Cheng
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  4. Dong Qing Liu
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Correspondence to Xue Lian Zheng.

Electronic supplementary material

12010_2015_1826_Fig6_ESM.ppt

Supplement Figure. 1 PCR analysis of Gus gene from genomic DNA of the sterile transgenic plantlets sub-cultured for 6 times (a) and cultured for 6 months in soil after a 3-time sub-culture (b) separately. M, DNA marker 2000; 25, 26, 27 transgenic lines induced by A. tumefaciens harboring pCAMBIA 3301; P, binary vector pCAMBIA 3301; WT, DNA of the normal S. miltiorrhiza. (PPT 251 kb)

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Liu, Y., Yang, S.X., Cheng, Y. et al. Production of Herbicide-Resistant Medicinal Plant Salvia miltiorrhiza Transformed with the Bar Gene. Appl Biochem Biotechnol 177, 1456–1465 (2015). https://doi.org/10.1007/s12010-015-1826-5

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  • DOI: https://doi.org/10.1007/s12010-015-1826-5

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