biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 49:361-370, 2005 | DOI: 10.1007/s10535-005-0009-6

Agrobacterium-mediated transformation of Pisum sativum in vitro and in vivo

L. ©vábová1, P. Smýkal1, M. Griga1,*, V. Ondřej1
1 AGRITEC Ltd., Plant Biotechnology Department, ©umperk, Czech Republic

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm-3 kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T0 putative transformants and their seed progenies (T1 to T3 generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T0 - T3 plants were morphologically normal and fertile.

Keywords: grain legumes; pea; transgene integration; transgene stability
Subjects: Agrobacterium tumefaciens; auxins; cultivar and genotype differences, β-glucuronidase; β-glucuronidase; nutrient medium B5 (Gamborg); nutrient medium Langley and Kado; nutrient medium MS (Murashige and Skoog); pea, transgenic plants; Pisum sativum; polymerase chain reaction (PCR); Southern blot analysis; transgene integration, transgene stability; transgenic plants

Received: July 9, 2004; Accepted: January 5, 2005; Published: September 1, 2005  Show citation

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©vábová, L., Smýkal, P., Griga, M., & Ondřej, V. (2005). Agrobacterium-mediated transformation of Pisum sativum in vitro and in vivo. Biologia plantarum, 49(3), 361-370. doi: 10.1007/s10535-005-0009-6
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    References

    1. Bean, S., Gooding, P.S., Mullineaux, P.M., Davies, D.R.: A simple system for pea transformation.-Plant Cell Rep. 16: 513-519, 1997. Go to original source...
    2. Bevan, M.: Binary Agrobacterium vectors for plant transformation.-Nucl. Acids Res. 12: 8711-8721, 1984. Go to original source...
    3. Bhagarva, S.C., Smigocki, A.C.: Transformation of tropical grain legumes using particle bombardment.-Curr. Sci. 66: 439-442, 1994.
    4. Boase, M.R., Butler, R.C., Borst, N.K.: Chrysanthemum cultivar-Agrobacterium interactions revealed by GUS expression time course experiments.-Sci. Hort. 77: 89-107, 1998. Go to original source...
    5. Charity, J.A., Anderson, M.A., Bittisnich, D.J., Whitecross, M., Higgins, T.J.V.: Transgenic tobacco and peas expressing a proteinase inhibitor from Nicotiana alata have increased insect resistance.-Mol. Breed. 5: 357-365, 1999. Go to original source...
    6. Chowrira, G.M., Akella, V., Lurquin, P.F.: Electroporation-mediated gene transfer into intact nodal meristems in planta: generating transgenic plants without in vitro tissue culture.-Mol. Biotechnol. 3: 17-23, 1995. Go to original source...
    7. Chowrira, G.M., Cavileer, T.D., Gupta, S.K., Lurquin, P.F., Berger, P.H.: Coat protein resistance to pea enation mosaic virus in transgenic Pisum sativum L.-Transgenic Res. 7: 265-271, 1998. Go to original source...
    8. Davies, D.R., Hamilton, J., Mullineaux, P.: Transformation of peas.-Plant Cell Rep. 12: 180-183, 1993. Go to original source...
    9. Davies, D.R., Mullineaux, P.M.: Tissue culture and transformation.-In: Casey, R., Davies, D.R. (ed.): Peas: Genetics, Molecular Biology and Biotechnology. Pp. 291-301. CAB International, Wallingford 1993.
    10. De Kathen, A., Jacobsen, H.-J.: Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors.-Plant Cell Rep. 9: 276-279, 1990. Go to original source...
    11. De Kathen, A., Jacobsen, H.-J.: Transformation of pea (Pisum sativum L.).-In: Bajaj, Y.P.S. (ed.): Biotechnology in Agriculture and Forestry. Vol. 23: Plant Protoplasts and Genetic Engineering IV. Pp. 331-347. Springer-Verlag, Berlin 1993. Go to original source...
    12. Doyle, J.J., Doyle, J.L.: A rapid DNA isolation procedure for small quantities of fresh leaf tissue.-Phytochem. Bull. 19: 11-15, 1987.
    13. Filippone, E., Lurquin, P.F.: Stable transformation of pea tissues after co-cultivation with two Agrobacterium tumefaciens strains.-Pisum Newslett. 21: 16-18, 1989.
    14. Forsbach, A, Schubert, D, Lechtenberg, B, Gils, M, Schmidt, R.: A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome.-Plant mol. Biol. 52: 161-76, 2003. Go to original source...
    15. Frisch, D.A., Harris-Haller, L.W., Yokubaitis, N.T., Thomas, T.L., Hardin, S.H., Hall, T.C.: Complete sequence of the binary vector Bin 19.-Plant mol. Biol. 27: 405-409, 1995. Go to original source...
    16. Futterer, J., Gisel, A., Iglesias, V., Kloti, A., Kost, B., Mittelsten Scheid, O., Neuhaus, G., Neuhaus-Url, G., Schrott, M., Shillito, R., Spangenberg, G., Wang, Z.Y.: Standard molecular techniques for the analysis of transgenic plants.-In: Potrykus, I., Spangenberg, G. (ed.): Gene Transfer to Plants, Springer Lab Manual. Pp. 215-263. Springer-Verlag, Berlin 1995. Go to original source...
    17. Gamborg, O.L., Miller, R.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells.-Exp. Cell Res. 50: 151-158, 1968. Go to original source...
    18. Grant, J.E., Cooper, P.A.: Genetic transformation in pea.-In: Jaiwal, P.K., Singh, R.P. (ed.): Applied Genetics of Leguminosae Biotechnology. Pp. 23-34. Kluwer Academic Publishers, Dordrecht 2003. Go to original source...
    19. Grant, J.E., Cooper, P.A., McAra, A.E., Frew, T.J.: Transformation of peas (Pisum sativum L.) using immature cotyledons.-Plant Cell Rep. 15: 254-258, 1995. Go to original source...
    20. Griga, M., Tejklova, E., Novak, F.J., Kubalakova, M.: In vitro clonal propagation of Pisum sativum L.-Plant Cell Tissue Organ Cult. 6: 95-104, 1986. Go to original source...
    21. Ho, M.W., Ryan, A., Cummins, J.: Hazards of transgenic plants containing the cauliflower mosaic viral promoter.-Microbiol. Ecol. Health Disease 12: 6-11, 2000. Go to original source...
    22. Hobbs, S.L.A., Jackson, J.A., Mahon, J.D.: Specificity of strain and genotype in the susceptibility of pea to Agrobacterium tumefaciens.-Plant Cell Rep. 8: 274-277, 1989. Go to original source...
    23. Hodal, L., Bochardt, A., Nielsen, J.E., Mattsson, O., Okkels, F.T.: Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and nontransgenic plants.-Plant Sci. 87: 115-122, 1992. Go to original source...
    24. Hood, E.E., Gelvin, S.B., Melchers, L.S., Hoekema, A.: New Agrobacterium helper plasmids for gene transfer to plants.-Transgenic Res. 2: 208-218, 1993. Go to original source...
    25. Hussey, G., Johnson, R.D., Warren, S.: Transformation of meristematic cells in the shoot apex of cultured pea shoots by Agrobacterium tumefaciens and A. rhizogenes.-Protoplasma 148: 101-105, 1989. Go to original source...
    26. Jackson, J.A., Hobbs, S.L.A.: Rapid multiple shoot production from cotyledonary node explants of pea (Pisum sativum L.).-In Vitro cell. dev. Biol. Plant 26: 835-838, 1990. Go to original source...
    27. Jaiwal, P.W., Kumari, R., Ignacimuthu, S., Potrykus, I., Sautter, Ch.: Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) - a recalcitrant grain legume.-Plant Sci. 161: 239-247, 2001. Go to original source...
    28. Jones, A.L., Johansen, I.E., Bean, S.J., Bach, I., Maule, A.J.: Specifity of resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (Nib) gene.-J. gen. Virol. 79: 3129-3137, 1998. Go to original source...
    29. Kaminski, P.A., Buffard, D., Strosberg, A.D.: The pea lectin gene family contains only one functional gene.-Plant mol. Biol. 9: 497-507, 1987. Go to original source...
    30. Kim, S.R, Lee, J., Jun, S.H., Park, S., Kang, H.G., Kwon, S., An, G.: Transgene structures in T-DNA-inserted rice plants.-Plant mol. Biol. 52: 761-73, 2003. Go to original source...
    31. Kohli, A., Griffiths, S., Palacios, N., Twyman, R.M., Vain, P., Laurie, D..A, Christou, p.: Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination.-Plant J. 17: 591-601, 1999. Go to original source...
    32. Krasnyanski, S.F., Sandhu, J., Domier, L.L., Buetow, D.E., Korban, S.S.: Effect of an enhanced CAMV 35S promoter and fruit-specific promoter on uidA gene expression in transgenic tomato plants.-In Vitro cell. dev. Biol. Plant 37: 427-433, 2001. Go to original source...
    33. Kumar, S., Fladung, M.: Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration.-Plant J. 31: 543-51, 2002. Go to original source...
    34. Kurkdjian, A., Manigault, P., Beardsley, R.: Crown gall: effect of temperature on tumorigenesis in pea seedlings.-Can. J. Bot. 47: 803-808, 1968. Go to original source...
    35. Langley, R.A., Kado, C.I.: Studies on Agrobacterium tumefaciens: conditions for mutagenesis by N-methyl-N-nitrosoquanidine and relationship of A. tumefaciens mutants to crown gall induction.-Mutat. Res. 14: 277-286, 1972. Go to original source...
    36. Lutova, L.A., Sharova, N.V.: [Variation in genetic forms of pea (Pisum sativum L.) in capacity for transformation with different strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes.]-Genetika (Moscow) 29: 1656-1671, 1993. [In Russ.]
    37. Malysheva, N.V., Pavlova, Z.B., Chernysh, N.S., Kravchenko, L.V., Kislin, Y.N., Chmelev, V., Lutova, L.A.: Genetic transformation of pea (Pisum sativum).-In: Bajaj, Y.P.S. (ed.): Biotechnology in Agriculture and Forestry. Vol. 47: Transgenic Crops II. Pp. 284-304. Springer-Verlag, Berlin 2001. Go to original source...
    38. Manigault, P., Kurkdjian, A.: Influence de la lumiere sur developpement des tumeurs de la plantule de pois Pisum sativum L., induites par Agrobacterium tumefaciens (Smith et Town) Conn.-Compt. rend. Acad. Sci. (Paris) 264: 2304-2306, 1967.
    39. Matzke, M.A, Mette, M.F., Aufsatz W.: More on CaMV.-Natur. Biotechnol. 18: 579, 2000. Go to original source...
    40. Morton, R.L., Gollasch, S., Schroeder, H.E., Bateman, K.S., Higgins, T.J.: Genetic technology in peas for improved field performance and enhanced grain quality.-In: Khachatourians, G., McHughen, A., Scorza, R., Nip, W.-K., Hui, Y.H. (ed.): Transgenic Plants and Crops. Pp. 197-205. Marcel Dekker, New York 2002.
    41. Morton, R.L., Schroeder, H.E., Bateman, K.S., Chrispeels, M.J., Armstrong, E., Higgins, T.J.V.: Bean α-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions.-Proc. nat. Acad. Sci. USA 97: 3820-3825, 2000. Go to original source...
    42. Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassay with tobacco tissue cultures.-Physiol. Plant. 15: 472-497, 1962. Go to original source...
    43. Nadolska-Orczyk, A., Orczyk, W.: Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.).-Mol. Breed. 6: 185-194, 2000. Go to original source...
    44. Nicolas, M., Gregoire, S., Teoule, E.: Gene transfer in pea (Pisum sativum L.) via somatic embryogenesis.-In: Proc. 2nd Europ. Conf. Grain Legumes. Pp. 424-425. AEP Publ., Paris 1995.
    45. Olhoft, P.M., Flagel, L.E., Donovan, C.M., Somers, D.A.: Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method.-Planta 216: 723-735, 2003. Go to original source...
    46. Polowick, P.L., Quandt, J., Mahon, J.D.: The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions.-Plant Sci. 153: 161-170, 2000. Go to original source...
    47. Puonti-Kaerlas, J., Eriksson, T., Engstrom, P: Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens-mediated gene transfer.-Theor. appl. Genet. 80: 246-252, 1990. Go to original source...
    48. Puonti-Kaerlas, J., Eriksson, T., Engstrom, P.: Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants.-Theor. appl. Genet. 84: 443-450, 1992. Go to original source...
    49. Puonti-Kaerlas, J., Stabel, P., Eriksson, T.: Transformation of pea (Pisum sativum L.) by Agrobacterium tumefaciens.-Plant Cell Rep. 8: 321-324, 1989. Go to original source...
    50. Qin, H., Dong, Y., Von Arnim, A.G.: Epigenetic interactions between Arabidopsis transgenes: characterization in light of transgene integration sites.-Plant mol. Biol. 52: 217-31, 2003. Go to original source...
    51. Sambrook, J., Fritsch, E.F., Maniatis, T.: Molecular Cloning: A Laboratory Manual, 2nd Edition.-Cold Spring Harbor Laboratory Press, New York 1989.
    52. Schaerer, S., Pilet, P.-E.: Roots, explants and protoplasts from pea transformed with strains of Agrobacterium tumefaciens and rhizogenes.-Plant Sci. 78: 247-258, 1991. Go to original source...
    53. Schroeder, H.E., Schotz, A.H., Wardley-Richardson, T., Spencer, D., Higgins, T.J.V.: Transformation and regeneration of two cultivars of pea (Pisum sativum L.).-Plant Physiol. 101: 751-757, 1993. Go to original source...
    54. Shade, R.E., Schroeder, H.E., Pueyo, J.J., Tabe, L.M., Murdock, L.L., Higgins, TJ.V., Chrispeels, M. J.: Transgenic pea seeds expressing the α-amylase inhibitor of the common bean are resistant to bruchid beetles.-Bio/Technology 12: 793-795, 1994. Go to original source...
    55. Shao, C.Y., Russinova, E., Iantcheva, A., Atanassov, A., McCormac, A., Chen, D.F., Elliott, M.C., Slater, A.: Rapid transformation and regeneration of alfalfa (Medicago falcata L.) via direct somatic embryogenesis.-Plant Growth Regul. 31: 155-166, 2000. Go to original source...
    56. Timmerman-Vaughan, G.M., Pither-Joyce, M.D., Cooper, P.A., Russell, A.C., Grant, J.E.: Partial resistance of transgenic peas to alfalfa mosaic virus under greenhouse and field conditions.-Crop Sci. 41: 846-853, 2001. Go to original source...
    57. Vain, P., James, A., Worland, B., Snape, W.: Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment.-Theor. appl. Genet. 105: 878-889, 2002. Go to original source...
    58. Vancanneyt, G., Schmidt, 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. Go to original source...
    59. Van Wordragen, M.F., De Jong, J., Schornagel, M.J., Dons, H.J.M.: Rapid screening for host-bacterium interactions in Agrobacterium-mediated gene transfer to chrysanthemum, by using the GUS-intron gene.-Plant Sci. 81: 207-214, 1992. Go to original source...
    60. Warkentin, T.D., Jordan, M.C., Hobbs, S.L.A.: Effect of promoter-leader sequences on transient reporter gene expression in particle bombarded pea (Pisum sativum L.) tissues.-Plant Sci. 87: 171-177, 1992. Go to original source...
    61. Wozniak, C.A., Owens, L.D.: Native β-glucuronidase activity in sugar beet (Beta vulgaris).-Physiol. Plant. 90: 763-771, 1994. Go to original source...

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