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Agrobacterium-Mediated Transformation of Switchgrass and Inheritance of the Transgenes

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Abstract

Switchgrass (Panicum virgatum L.) has been developed into an important biofuel crop. Embryogenic calli induced from caryopses or inflorescences of the lowland switchgrass cultivar Alamo were used for Agrobacterium-mediated transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin as the selection agent. Embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105. Calli resistant to hygromycin were obtained after 5 to 8 weeks of selection. Soil-grown transgenic switchgrass plants were obtained 4 to 5 months after Agrobacterium infection. The transgenic nature of the regenerated plants was demonstrated by PCR, Southern blot hybridization analysis, and GUS staining. T1 progeny were obtained after reciprocal crosses between transgenic and untransformed control plants. Molecular analyses of the T1 progeny revealed various patterns of segregation. Transgene silencing was observed in the progeny with multiple inserts. Interestingly, reversal of the expression of the silenced transgene was found in segregating progeny with a single insert.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BAP:

6-Benzylaminopurine

PCR:

Polymerase chain reaction

References

  1. US Department of Energy (2006) Breaking the biological barriers to cellulosic ethanol: a joint research agenda. DOE/SC-0095

  2. Bouton JH (2007) Molecular breeding of switchgrass for use as a biofuel crop. Curr Opin Genet Dev 17:553–558

    Article  CAS  PubMed  Google Scholar 

  3. Gressel J (2008) Transgenics are imperative for biofuel crops. Plant Sci 174:246–263

    Article  CAS  Google Scholar 

  4. McLaughlin SB, Kiniry JR, Taliaferro CM, De La Torre Ugarte D, Donald LS (2006) Projecting yield and utilization potential of switchgrass as an energy crop. Adv Agron 90:267–297

    Article  Google Scholar 

  5. Wang Z-Y, Ge Y (2006) Recent advances in genetic transformation of forage and turf grasses. In Vitro Cell Dev Biol, Plant 42:1–18

    Google Scholar 

  6. Cheng M, Lowe BA, Spencer TM, Ye XD, Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev Biol, Plant 40:31–45

    Article  Google Scholar 

  7. Richards HA, Rudas VA, Sun H, McDaniel JK, Tomaszewski Z, Conger BV (2001) Construction of a GFP-BAR plasmid and its use for switchgrass transformation. Plant Cell Rep 20:48–54

    Article  CAS  Google Scholar 

  8. Somleva MN, Tomaszewski Z, Conger BV (2002) Agrobacterium-mediated genetic transformation of switchgrass. Crop Sci 42:2080–2087

    Article  CAS  Google Scholar 

  9. Somleva M, Snell K, Beaulieu J, Peoples O, Garrison B, Patterson N (2008) Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop. Plant Biotechnol J 6:663–678

    Article  CAS  PubMed  Google Scholar 

  10. Dixon RA, Bouton JH, Narasimhamoorthy B, Saha M, Wang Z-Y, May GD (2007) Beyond structural genomics for plant science. Adv Agron 95:77–161

    Article  CAS  Google Scholar 

  11. Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892–899

    Article  CAS  PubMed  Google Scholar 

  12. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  13. Moore KJ, Moser LE, Vogel KP, Waller SS, Johnson BE, Pedersen JF (1991) Describing and quantifying growth stages of perennial forage grasses. Agron J 83:1073–1077

    Google Scholar 

  14. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    Article  CAS  Google Scholar 

  15. Nandakumar R, Chen L, Rogers SMD (2004) Factors affecting the Agrobacterium-mediated transient transformation of the wetland monocot, Typha latifolia. Plant Cell, Tissue Organ Cult 79:31–38

    Article  CAS  Google Scholar 

  16. Doyle JJ, DJ L (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  17. Mendel RR, Mueller B, Schulze J, Kolesnikov V, Zelenin A (1989) Delivery of foreign genes to intact barley cells by high-velocity microprojectiles. Theor Appl Genet 78:31–34

    Article  CAS  Google Scholar 

  18. Xiao K, Zhang C, Harrison M, Wang Z-Y (2005) Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants. Mol Breed 15:221–231

    Article  CAS  Google Scholar 

  19. Wang Z-Y, Bell J, Ge YX, Lehmann D (2003) Inheritance of transgenes in transgenic tall fescue (Festuca arundinacea Schreb.). In Vitro Cell Dev Biol Plant 39:277–282

    Article  CAS  Google Scholar 

  20. Wang Z-Y, Ge Y (2005) Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea Schreb.). J Plant Physiol 162:103–113

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. Spangenberg G, Wang Z-Y, Wu XL, Nagel J, Potrykus I (1995) Transgenic perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells. Plant Sci 108:209–217

    Article  CAS  Google Scholar 

  23. Wang Z-Y, Ge Y (2005) Rapid and efficient production of transgenic bermudagrass and creeping bentgrass bypassing the callus formation phase. Funct Plant Biol 32:769–776

    Article  CAS  Google Scholar 

  24. Spangenberg G, Wang Z-Y, Potrykus I (1998) Biotechnology in forage and turf grass improvement. Springer, Berlin

    Google Scholar 

  25. Alexandrova KS, Denchev PD, Conger BV (1996) Micropropagation of switchgrass by node culture. Crop Sci 36:1709–1711

    CAS  PubMed  Google Scholar 

  26. Lechtenberg B, Schubert D, Forsbach A, Gils M, Schmidt R (2003) Neither inverted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing. Plant J 34:507–517

    Article  CAS  PubMed  Google Scholar 

  27. Fagard M, Vaucheret H (2003) (Trans)Gene silencing in plants: how many mechanisms? Annu Rev Plant Physiol Plant Mol Biol 51:167–194

    Article  Google Scholar 

  28. Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 58:545–554

    Article  CAS  PubMed  Google Scholar 

  29. Anand A, Trick HN, Gill BS, Muthukrishnan S (2003) Stable transgene expression and random gene silencing in wheat. Plant Biotechnol J 1:241–251

    Article  CAS  PubMed  Google Scholar 

  30. Sticklen M (2006) Plant genetic engineering to improve biomass characteristics for biofuels. Curr Opin Biotechnol 17:315–319

    Article  CAS  PubMed  Google Scholar 

  31. Hisano H, Nandakumar R, Wang Z-Y (2009) Genetic modification of lignin biosynthesis for improved biofuel production. In Vitro Cell Dev Biol, Plant 45:306–313

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Frank Hardin and Jackie Kelly for critical reading of the manuscript. The work was supported by the US Department of Agriculture and US Department of Energy Biomass Initiative (project no. 2009-10003-05140), the BioEnergy Science Center, and the Samuel Roberts Noble Foundation. The BioEnergy Science Center is supported by the Office of Biological and Environmental Research in the DOE Office of Science. This report was prepared as an account of work partly sponsored by the US Government. Neither the US Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product or process disclosed or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the US Government or any agency thereof. The views and opinions of the authors expressed herein do not necessarily reflect those of the US Government or any agency thereof.

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Authors and Affiliations

  1. Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

    Yajun Xi, Chunxiang Fu, Yaxin Ge, Rangaraj Nandakumar, Hiroshi Hisano, Joseph Bouton & Zeng-Yu Wang

  2. College of Agriculture, Northwest A and F University, Yangling, Shaanxi, 712100, China

    Yajun Xi

  3. BioEnergy Science Center, Oak Ridge, TN, USA

    Rangaraj Nandakumar, Hiroshi Hisano & Zeng-Yu Wang

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  1. Yajun Xi
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  2. Chunxiang Fu
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  3. Yaxin Ge
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  4. Rangaraj Nandakumar
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  5. Hiroshi Hisano
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  6. Joseph Bouton
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  7. Zeng-Yu Wang
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Correspondence to Zeng-Yu Wang.

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Yajun Xi and Chunxiang Fu contributed equally to this work.

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ESM

Fig. 1. a PCR analysis of T1 progeny derived from the transgenic plant TB8-8-1. b Southern blot hybridization analysis of DNA samples from the transgenic plant TB8-8-1 and its T1 progeny. Hybridization probe: hph probe. Fig. 2. Integration and expression of the hph transgene in the transgenic plant TB8-8-40 and its progeny. a Southern hybridization of a DNA blot containing HindIII digested genomic DNA isolated from the TB8-8-40 transgenic plant and its T1 progeny. b RT-PCR analysis of hph transcriptional level in the TB8-8-40 plant and its T1 progeny. c hygromycin resistance of leaves from TB8-8-40 and its progeny (PPTX 3122 kb)

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Xi, Y., Fu, C., Ge, Y. et al. Agrobacterium-Mediated Transformation of Switchgrass and Inheritance of the Transgenes. Bioenerg. Res. 2, 275–283 (2009). https://doi.org/10.1007/s12155-009-9049-7

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