Agrobacterium-Mediated Transformation of Cotton

Part of the Methods in Molecular Biology book series (MIMB, volume 958)


There are many methods and techniques that can be used to transfer foreign genes into cells. In plant biotechnology, Agrobacterium-mediated transformation is a widely used traditional method for inserting foreign genes into plant genome and obtaining transgenic plants, particularly for dicot plant species. Agrobacterium-mediated transformation of cotton involves several important and also critical steps, which includes coculture of cotton explants with Agrobacterium, induction and selection of stable transgenic cell lines, recovery of plants from transgenic cells majorly through somatic embryogenesis, and detection and expression analysis of transgenic plants. In this chapter, we describe a detailed step-by-step protocol for obtaining transgenic cotton plants via Agrobacterium-mediated transformation.


  1. 1.
    Birch RG (1997) Plant transformation: problems and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol 48:297–326PubMedCrossRefGoogle Scholar
  2. 2.
    Gelvin SB (2003) Agobacterium-mediated plant transformation: the biology behind the “gene-Jockeying” tool. Microbiol Mol Biol Rev 67:16–37PubMedCrossRefGoogle Scholar
  3. 3.
    Firoozabady E, Deboer DL, Merlo DJ, Halk EL, Amerson LN, Rashka KE, Murray EE (1987) Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol Biol 10:105–116CrossRefGoogle Scholar
  4. 4.
    Umbeck P, Johnson G, Barton K, Swain W (1987) Genetically transformed cotton (Gossypium hirsutum L.) plants. Bio-Technology 5:263–266Google Scholar
  5. 5.
    Asad S, Mukhtar Z, Nazir F, Hashmi JA, Mansoor S, Zafar Y, Arshad M (2008) Silicon carbide whisker-mediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants. Mol Biotechnol 40:161–169PubMedCrossRefGoogle Scholar
  6. 6.
    Chen TZ, Wu SJ, Zhao J, Guo WZ, Zhang TZ (2010) Pistil drip following pollination: a simple in planta Agrobacterium-mediated transformation in cotton. Biotechnol Lett 32:547–555CrossRefGoogle Scholar
  7. 7.
    Hashmi JA, Zafar Y, Arshad M, Mansoor S, Asad S (2011) Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences. Virus Genes 42:286–296PubMedCrossRefGoogle Scholar
  8. 8.
    Katageri IS, Vamadevaiah HM, Udikeri SS, Khadi BM, Kumar PA (2007) Genetic transformation of an elite Indian genotype of cotton (Gossypium hirsutum L.) for insect resistance. Curr Sci 93:1843–1847Google Scholar
  9. 9.
    Kim HJ, Murai N, Fang DD, Triplett BA (2009) Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues. Plant Sci 180:323–332CrossRefGoogle Scholar
  10. 10.
    Li FF, Wu SJ, Chen TZ, Zhang J, Wang HH, Guo WZ, Zhang TZ (2009) Agrobacterium-mediated co-transformation of multiple genes in upland cotton. Plant Cell Tissue Organ Cult 97:225–235CrossRefGoogle Scholar
  11. 11.
    Liu JF, Zhao CY, Ma J, Zhang GY, Li MG, Yan GJ, Wang XF, Ma ZY (2009) Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) with a fungal phytase gene improves phosphorus acquisition. Euphytica 181:31–40CrossRefGoogle Scholar
  12. 12.
    Nandeshwar SB, Moghe S, Chakrabarty PK, Deshattiwar MK, Kranthi K, Anandkumar P, Mayee CD, Khadi BM (2009) Agrobacterium-mediated transformation of cry1Ac gene into shoot-tip meristem of diploid cotton Gossypium arboreum cv. RG8 and regeneration of transgenic plants. Plant Mol Biol Rep 27:549–557CrossRefGoogle Scholar
  13. 13.
    Wu JH, Luo XL, Zhang XR, Shi YJ, Tian YC (2011) Development of insect-resistant ­transgenic cotton with chimeric TVip3A*accumulating in chloroplasts. Transgenic Res 20:963–973PubMedCrossRefGoogle Scholar
  14. 14.
    Wu SJ, Wang HH, Li FF, Chen TZ, Zhang J, Jiang YJ, Ding YZ, Guo WZ, Zhang TZ (2008) Enhanced Agrobacterium-mediated transformation of embryogenic calli of upland cotton via efficient selection and timely subculture of somatic embryos. Plant Mol Biol Rep 26:174–185CrossRefGoogle Scholar
  15. 15.
    Zhang J, Cai L, Cheng JQ, Mao HZ, Fan XP, Meng ZH, Chan KM, Zhang HJ, Qi JF, Ji LH, Hong Y (2008) Transgene integration and organization in cotton (Gossypium hirsutum L.) genome. Transgenic Res 17:293–306PubMedCrossRefGoogle Scholar
  16. 16.
    Ikram Ul H (2004) Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) via vacuum infiltration. Plant Mol Biol Rep 22:279–288CrossRefGoogle Scholar
  17. 17.
    Leelavathi S, Sunnichan VG, Kumria R, Vijaykanth GP, Bhatnagar RK, Reddy VS (2004) A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22:465–470PubMedCrossRefGoogle Scholar
  18. 18.
    Satyavathi VV, Prasad V, Lakshmi BG, Sita GL (2002) High efficiency transformation protocol for three Indian cotton varieties via Agrobacterium tumefaciens. Plant Sci 162:215–223CrossRefGoogle Scholar
  19. 19.
    Sunilkumar G, Rathore KS (2001) Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Mol Breed 8:37–52CrossRefGoogle Scholar
  20. 20.
    Tohidfar M, Mohammadi M, Ghareyazie B (2005) Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) using a heterologous bean chitinase gene. Plant Cell Tissue Organ Cult 83:83–96CrossRefGoogle Scholar
  21. 21.
    Yuceer SU, Koc NK (2006) Agrobacterium-mediated transformation and regeneration of cotton plants. Russian J Plant Physiol 53:413–417CrossRefGoogle Scholar
  22. 22.
    Zhao FY, Li YF, Xu PL (2006) Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L. cv. Zhongmian 35) using glyphosate as a selectable marker. Biotechnol Lett 28:1199–1207PubMedCrossRefGoogle Scholar
  23. 23.
    Zhu SW, Gao P, Sun JS, Wang HH, Luo XM, Jiao MY, Wang ZY, Xia GX (2006) Genetic transformation of green-colored cotton. Vitro Cell Dev Biol Plant 42:439–444CrossRefGoogle Scholar
  24. 24.
    Murashige T, Skoog F (1962) A fdvised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  25. 25.
    Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158PubMedCrossRefGoogle Scholar
  26. 26.
    Jin SX, Zhang XL, Liang SG, Nie YC, Guo XP, Huang C (2005) Factors affecting transformation efficiency of embryogenic callus of upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 81:229–237CrossRefGoogle Scholar
  27. 27.
    Joubert P, Beaupere D, Lelievre P, Wadouachi A, Sangwan RS, Sangwan-Norreel BS (2002) Effects of phenolic compounds on Agrobacterium vir genes and gene transfer induction – a plausible molecular mechanism of phenol binding protein activation. Plant Sci 162:733–743CrossRefGoogle Scholar
  28. 28.
    Lai E-M, Shih H-W, Wen S-R, Cheng M-W, Hwang H-H, Chiu S-H (2006) Proteomic analysis of Agrobacterium tumefaciens response to the vir gene inducer acetosyringone. Proteomics 6:4130–4136PubMedCrossRefGoogle Scholar
  29. 29.
    Nair GR, Lai X, Wise AA, Rhee BW, Jacobs M, Binns AN (2011) The integrity of the periplasmic domain of the VirA sensor kinase is critical for optimal coordination of the virulence signal response in Agrobacterium tumefaciens. J Bacteriol 193:1436–1448PubMedCrossRefGoogle Scholar
  30. 30.
    Stachel SE, Messens E, Vanmontagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629CrossRefGoogle Scholar
  31. 31.
    Aggarwal D, Kumar A, Reddy MS (2011) Agrobacterium tumefaciens mediated genetic transformation of selected elite clones of Eucalyptus tereticornis. Acta Physiol Plant 33:1603–1611CrossRefGoogle Scholar
  32. 32.
    Bhuiyan MSU, Min SR, Jeong WJ, Sultana S, Choi KS, Lim YP, Song WY, Lee Y, Liu JR (2011) An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea. Plant Biotechnol 28:17–23CrossRefGoogle Scholar
  33. 33.
    Mehrotra M, Sanyal I, Amla DV (2011) High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants. Plant Cell Rep 30:1603–1616PubMedCrossRefGoogle Scholar
  34. 34.
    Rashid H, Chaudhry Z, Khan MH (2011) Effect of explant plant source and acetosyringone concentration on transformation efficiency of wheat cultivars. Afr J Biotechnol 10:8737–8740Google Scholar
  35. 35.
    Wu S-J, Wang H-H, Li F-F, Chen T-Z, Zhang J, Jiang Y-J, Ding Y, Guo W-Z, Zhang T-Z (2008) Enhanced Agrobacterium-mediated transformation of embryogenic calli of upland cotton via efficient selection and timely subculture of somatic embryos. Plant Mol Biol Rep 26:174–185CrossRefGoogle Scholar
  36. 36.
    Dillen W, De Clercq J, Kapila J, Zambre M, Van Montagu M, Angenon G (1997) The effect of temperature on Agrobacterium tumefaciens-mediated gene transfer to plants. Plant J 12:1459–1463CrossRefGoogle Scholar
  37. 37.
    Uranbey S, Sevimay CS, Kaya MD, Ipek A, Sancak C, Basalma D, Er C, Ozcan S (2005) Influence of different co-cultivation temperatures, periods and media on Agrobacterium tumefaciens-mediated gene transfer. Biol Plant 49:53–57CrossRefGoogle Scholar
  38. 38.
    Wang B, Liu L, Wang X, Yang J, Sun Z, Zhang N, Gao S, Xing X, Peng D (2009) Transgenic ramie [Boehmeria nivea (L.) Gaud.]: factors affecting the efficiency of Agrobacterium tumefaciens-mediated transformation and regeneration. Plant Cell Rep 28:1319–1327PubMedCrossRefGoogle Scholar
  39. 39.
    Zhang B-H, Liu F, Liu Z-H, Wang H-M, Yao C-B (2001) Effects of kanamycin on tissue culture and somatic embryogenesis in cotton. Plant Growth Regul 33:137–149CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of BiologyEast Carolina UniversityGreenvilleUSA

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