Minor modifications in obtainable Arabidopsis floral dip method enhances transformation efficiency and production of homozygous transgenic lines harboring a single copy of transgene

Priyanka Das, Naveen Chandra Joshi

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DOI: 10.4236/abb.2011.22010                

Abstract

Many researchers have developed various methods for in-planta or floral dip transformation of Arabidopsis thaliana, one of the simple protocol and widely used to produce transgenic Arabidopsis. As the efficiency and ease of getting a transformant is very much time consuming effort and less number of the transformants people get, we have developed a little modified transformation protocol to avoid the disparities. Four types of inoculums (inoculum1, inoculum2, inoculum3 and inoculum4) were used to check the transformation efficiency out of which Inoculum3 showed the highest rate of transformation among the four types. 0.07% Twin-20 also acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis. After vacuum infiltration keeping the Agrobacterium infected plants for 7-8 hrs horizontally in low light at 280C temperature condition, considered best to get an increased number of transformed seeds. Modified protocol produced ~12-14% increase in transformants. Selection pots (kanamycin supplemented soil filled pots) in place of selection plates (Kanamycin supplemented Murashige and Skoog agar plates) proved beneficial as no MS medium and no aseptic condition is required for selection of transformed plants. This increase in transformation efficiency consequently increased the percentage of homozygous and single copied stable transgenic lines.

Keywords

Arabidopsis thaliana; Floral-Dipping; Copy Number; Homozygous; Inoculum; Transgenic Plant

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Feldmann, K.A. and Marks, M.D. (1987) Agrobacterium mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach. Molecular and General Genetics, 208, 1-9. doi:10.1007/BF00330414
[2]	Bechtold, N., Ellis, J. and Pelletier, G. (1993) In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. Comptes Rendus de l Academie des Sciences Paris, Sciences de la vie/Life Sciences, 316, 1194-1199.
[3]	Bechtold, N., Jolivet. S., Voisin, R. and Pelletier, G. (2003) The endosperm and the embryo of Arabidopsis thaliana are independently transformed through infiltration by Agrobacterium tumefaciens. Transgenic Research, 12, 509-517. doi:10.1023/A:1024272023966
[4]	Clough, S.J. and Bent, A.F. (1998) Floral dip: A simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. Plant Journal, 16, 735-743. doi:10.1046/j.1365-313x.1998.00343.x
[5]	Desfeux, C., Clough, S. J. and Bent, A. F. (2000) Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiology, 123, 895-904. doi:10.1104/pp.123.3.895
[6]	Bechtold, N., Jolivet, S., Voisin, R. and Pelletier, G. (2003) The endosperm and the embryo of Arabidopsis thaliana are independently transformed through infiltration by Agrobacterium tumefaciens. Transgenic Research, 12, 509-517. doi:10.1023/A:1024272023966
[7]	Labra, M. et al. (2004) Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip. Theoretical and Applied Genetics, 109, 1512-1518. doi:10.1007/s00122-004-1773-y
[8]	Chung, M.H., Chen, M.K. and Pan, S.M. (2000) Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Reserch, 9, 471-476. doi:10.1023/A:1026522104478
[9]	Cao, M.Q., et al. (2000) Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration. Molecular Breeding, 6, 67-72. doi:10.1023/A:1009658128964
[10]	Tague, B.W. (2001) Germ-line transformation of Arabidopsis lasiocarpa. Transgenic Research, 10, 259-267. doi:10.1023/A:1016633617908
[11]	Wang, W.C., Menon, G. and Hansen, G. (2003) Development of a novel Agrobacterium mediated transformation method to recover transgenic Brassica napus plants. Plant Cell Report, 22, 274-281. doi:10.1007/s00299-003-0691-9
[12]	Harrison, S.J., Mott, E.K., Parsley, K., Aspinall, S., Gray, J.C. and Cottage, A. (2006) A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation. Plant Methods, 2, 19. doi:10.1186/1746-4811-2-19
[13]	Liu, N.Y., Zhang, Z.F. and Yang, W.C. (2008) Isolation of embryo-specific mutants in Arabidopsis: Plant transformation. Methods in Molecular Biology, 427, 91-100. doi:10.1007/978-1-59745-273-1_7
[14]	Logemann, E., Birkenbihl, R.P., Ulker, B. amd Somssich, I.E. (2006) An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods, 2, 16. doi:10.1186/1746-4811-2-16
[15]	Zhang, X., Henriques, R., Lin, S.S., Niu, Q.W. and Chua, N.H. (2006) Agrobacterium mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols, 1, 641-646. doi:10.1038/nprot.2006.97
[16]	Dellaporta, S.I., Wood, J. and Hicks, J.B. (1983) A plant DNA mini preparation: Version II. Plant molecular Biology Reporter, 1, 19-21. doi:10.1007/BF02712670
[17]	Fu, J., Momcilovic, I., Clemente, T.E., Nersesian, N., Trick, H.N. and Ristic, Z. (2008) Heterologous expression of a plastid EF-Tu reduces protein thermal eggregation and enhances CO2 fixation in wheat (Triticum aestivum) following exposure to heat stress. Plant molecular biology, 68, 277-288. doi:10.1007/s11103-008-9369-6
[18]	Miguel, M.T., Veronica, L.B., Joseluis, C.P. and Luis, H.E. (2004) Improving transformation efficiency of Arabidopsis thaliana by modifying the floral dip method. Plant Molecular Biology Reporter, 22, 63-70. doi:10.1007/BF02773350
[19]	Curtis, S.I. and Nam, G.H (2001) Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method-plant development and surfactant are important in optimizing transformation efficiency. Transgenic Research, 10, 363-371. doi:10.1023/A:1016600517293
[20]	Cough, J.S. (2005) Floral dip: Agobacterium-mediated germ line transformstion. Methods in Molecular Biology, 286, 91-102.
[21]	Davis, M.A., Hall, A., Millar, J.A., Darrah, C. and Davis, J.S. (2009) Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana. Plant Methods, 5, 3. doi:10.1186/1746-4811-5-3
[22]	Grevelding, C., Fantes, V., Kemper, E., Schell, J. and Masterson, R. (1993) Single copy T-DNA insertions in Arabidopsis are the predominant form of integration in root derived trnsgenics, where as multiple insertions are found in leaf discs. Plant Moleclar Biology, 23, 847-860. doi:10.1007/BF00021539
[23]	Lechtenberg, B., Schubert, D., Forsbach, A., Gils, M. and Schmidt, R. (2003) Inverted repeat T-DNA configuratios nor arrengements of trigger transgene scilencing. Plant Journal, 34, 507-517. doi:10.1046/j.1365-313X.2003.01746.x
[24]	Fu, J. and Ristic, Z. (2010) Analysis of transgenic wheat (Triticum aestivum L.) harboring a maize (Zea mays L.) gene for plastid EF-Tu: Segregation pattern, expression and effects of the transgene. Plant Molecular Biology, 73, 339-347. doi:10.1007/s11103-010-9622-7