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Biolistic-mediated genetic transformation of cowpea (Vigna unguiculata) and stable Mendelian inheritance of transgenes

  • Genetic Transformation and Hybridization
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Abstract

We describe a novel system of exploiting the biolistic process to generate stable transgenic cowpea (Vigna unguiculata) plants. The system is based on combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5′ regulatory sequence) and a simple tissue culture protocol. The gus gene (under control of the act2 promoter) was used as a reporter gene. The transformation frequency (defined as the total number of putative transgenic plants divided by the total number of embryonic axes bombarded) was 0.90%. Southern analyses showed the presence of both ahas and gus expression cassettes in all primary transgenic plants, and demonstrated one to three integrated copies of the transgenes into the genome. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion. Western blot analysis revealed that the GUS protein expressed in the transgenic plants had the same mass and isoelectric point as the bacterial native protein. This is the first report of biolistic-mediated cowpea transformation in which fertile transgenic plants transferred the foreign genes to next generations following Mendelian laws.

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References

  • Akella V, Lurquin PF (1993) Expression in cowpea seedlings of chimeric transgenes after electroporation into seed-derived embryos. Plant Cell Rep 12:110–117. doi:10.1007/BF00241945

    Article  CAS  Google Scholar 

  • Aliyu B (2007) Heritability and gene effects for incorporating pubescence into cowpea (Vigna unguiculata (L) Walp.) from V. rhomboidea Burtt. Davy. Euphytica 155:295–303. doi:10.1007/s10681-006-9331-0

    Article  Google Scholar 

  • Aragão FJL, Campos FAP (2007) Common bean and cowpea. In: Pua EC, Davey MR (eds) Biotechnology in agriculture and forestry. Transgenic crops IV. Springer, Berlin, pp 263–276

    Google Scholar 

  • Aragão FJL, Rech EL (1997) Morphological factors influencing recovery of transgenic bean plants (Phaseolus vulgaris L.) of a carioca cultivar. Int J Plant Sci 158:157–163. doi:10.1086/297426

    Article  Google Scholar 

  • Aragão FJL, Barros LMG, Brasileiro ACM, Ribeiro SG, Smith FD, Sanford JC, Faria JC, Rech EL (1996) Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theor Appl Genet 93:142–150. doi:10.1007/BF00225739

    Article  Google Scholar 

  • Aragão FJL, Sarokin L, Vianna GR, Rech EL (2000) Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean (Glycine max (L.) Merril) plants at high frequency. Theor Appl Genet 101:1–6. doi:10.1007/s001220051441

    Article  Google Scholar 

  • Aragão FJL, Vianna GR, Carvalheira SBRC, Rech EL (2005) Germ line genetic transformation in cotton (Gossypium hirsutum L.) by selection of transgenic meristematic cells with a herbicide molecule. Plant Sci 168:1227–1233. doi:10.1016/j.plantsci.2004.12.024

    Article  CAS  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Chaudhury D, Madanpotra S, Jaiwal R, Saini R, Kumar AP, Jaiwal PK (2007) Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L. Walp.) cultivar and transmission of transgenes into progeny. Plant Sci 172:692–700. doi:10.1016/j.plantsci.2006.11.009

    Article  CAS  Google Scholar 

  • Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349. doi:10.1093/nar/19.6.1349

    Article  PubMed  CAS  Google Scholar 

  • Fang J, Chao CT, Roberts PA, Ehlers JD (2007) Genetic diversity of cowpea [Vigna unguiculata (L.) Walp.] in four West African and USA breeding programs as determined by AFLP analysis. Genet Resour Crop Evol 54:1197–1209. doi:10.1007/s10722-006-9101-9

    Article  CAS  Google Scholar 

  • FAOSTAT (2006) FAO agriculture database. FAO, Rome. Available at http://faostat.fao.org/faostat

  • Garcia JA, Hille J, Goldbach R (1986) Transformation of cowpea (Vigna unguiculata) cells with an antibiotic-resistance gene using a Ti-plasmid-derived vector. Plant Sci 44:37–46. doi:10.1016/0168-9452(86)90166-4

    Article  CAS  Google Scholar 

  • Garcia JA, Hille J, Vos P, Goldbach R (1987) Transformation of cowpea (Vigna unguiculata) with a full-length DNA copy of cowpea mosaic virus M-RNA. Plant Sci 48:89–98. doi:10.1016/0168-9452(87)90135-X

    Article  CAS  Google Scholar 

  • Gomathinayagam P, Ganesh ram S, Rathnaswamy R, Ramaswamy NM (1998) Interspecific hybridization between Vigna unguiculata (L.) Walp. and V. vexillata (L.) A. Rich through in vitro embryo culture. Euphytica 102:203–209. doi:10.1023/A:1018381614098

    Article  Google Scholar 

  • Gulati A, Schryer P, Mchughen A (2002) Production of fertile transgenic lentil (Lens culinaris Medik) plants using particle bombardment. In vitro Cell Dev Biol Plant 38:316–324. doi:10.1079/IVP2002303

    Article  CAS  Google Scholar 

  • Ikea J, Ingelbrecht I, Uwaifo A, Thotttappilly G (2003) Stable gene transformation in cowpea (Vigna unguiculata L. Walp.) using particle gun method. Afr J Biotechnol 2:211–218

    CAS  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405. doi:10.1007/BF02667740

    Article  CAS  Google Scholar 

  • Murdock LL (1992) Improving insect resistance in cowpea through biotechnology. In: Thottappilly G, Monti LM, Mohan Raj DR, Moore AW (eds) Biotechnology: enhancing research on tropical crops in Africa. CTA-IITA, pp, pp 313–320

    Google Scholar 

  • Muthukumar B, Mariamma M, Veluthambi K, Gnanam A (1996) Genetic transformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens. Plant Cell Rep 15:980–985. doi:10.1007/BF00231600

    CAS  Google Scholar 

  • Obatolu VA (2003) Growth pattern of infants fed with a mixture of extruded malted maize and cowpea. Nutrition 19:174–178. doi:10.1016/S0899-9007(02)01102-4

    Article  PubMed  Google Scholar 

  • Penza R, Lurquin PF, Filippone E (1991) Gene transfer by co-cultivation of mature embryos with Agrobacterium tumefaciens—application to cowpea (Vigna unguiculata Walp). J Plant Physiol 138:39–43

    CAS  Google Scholar 

  • Penza R, Akella V, Lurquin PF (1992) Transient expression and histological localization of a gus chimeric gene after direct transfer to mature cowpea embryos. Biotechniques 13:576–580

    PubMed  CAS  Google Scholar 

  • Phillips RD, McWatters KH, Chinnan MS, Hung Y-C, Beuchat LR, Sefa-Dedeh S, Sakyi-Dawson S-D, Ngoddy P, Nnanyelugo D, Enwere J, Komey NS, Liu K, Mensa-Wilmot Y, Nnanna IA, Okeke C, Prinyawiwatkul W, Saalia FK (2003) Utilization of cowpeas for human food. Field Crops Res 82:193–213. doi:10.1016/S0378-4290(03)00038-8

    Article  Google Scholar 

  • Polowick PL, Quandt J, Mahon JD (2000) The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions. Plant Sci 153:161–170. doi:10.1016/S0168-9452(99)00267-8

    Article  PubMed  CAS  Google Scholar 

  • Popelka JC, Gollasch S, Moore A, Molvig L, Higgins TJV (2006) Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny. Plant Cell Rep 25:304–312. doi:10.1007/s00299-005-0053-x

    Article  PubMed  CAS  Google Scholar 

  • Rech EL, Vianna GR, Aragão FJL (2008) High efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat Protoc 3:410–418. doi:10.1038/nprot.2008.9

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA

    Google Scholar 

  • Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892–899. doi:10.1104/pp.102.017681

    Article  PubMed  CAS  Google Scholar 

  • Steel RGD, Torrie JH (1980) Principles and procedures of statistics. McGraw-Hill, New York

    Google Scholar 

  • Taiwo MA, Kareem KT, Nsa IY, Hughes JD (2007) Cowpea viruses: Effect of single and mixed infections on symptomatology and virus concentration. Virol J 4:95. doi:10.1186/1743-422X-4-95

    Article  PubMed  CAS  Google Scholar 

  • Vasconcelos EAR, Nogueira FCS, Abreu EFM, Gonçalves EF, Souza PAS, Campos FAP (2005) Protein extraction from cowpea tissues for 2D electrophoresis and MS analysis. Chromatographia 62:447–450. doi:10.1365/s10337-005-0637-1

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by Embrapa, Renorbio (Renorbio - Rede Nordeste de Biotecnologia) and CNPq/MCT. The authors would like to thank Dr. Francisco Rodrigues Freire Filho (Embrapa Meio Norte, Brazil) for providing the cowpea seeds and Dr. Michael Richardson for reading the manuscript.

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Authors and Affiliations

  1. Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 Norte, Asa Norte, Brasília, DF, 70770-200, Brazil

    Nayche L. Ivo, Cristina P. Nascimento, Lívia S. Vieira & Francisco J. L. Aragão

  2. Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Bloco 907, Fortaleza, CE, 60451-970, Brazil

    Francisco A. P. Campos

Authors
  1. Nayche L. Ivo
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  2. Cristina P. Nascimento
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  3. Lívia S. Vieira
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  4. Francisco A. P. Campos
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  5. Francisco J. L. Aragão
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Corresponding author

Correspondence to Francisco J. L. Aragão.

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Communicated by D. Somers.

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Ivo, N.L., Nascimento, C.P., Vieira, L.S. et al. Biolistic-mediated genetic transformation of cowpea (Vigna unguiculata) and stable Mendelian inheritance of transgenes. Plant Cell Rep 27, 1475–1483 (2008). https://doi.org/10.1007/s00299-008-0573-2

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  • DOI: https://doi.org/10.1007/s00299-008-0573-2

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