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Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions

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Summary

In order to introduce currently-available genes with agronomical value into banana, two genetic transformation protocols have been optimized.

Firstly, regenerable protoplasts isolated from embryogenic cell suspensions of the cultivar Bluggoe have been used for the introduction of several chimaeric uidA gene constructs by electroporation. With the inclusion of polyethylene glycol and heat shock, the frequency of transiently expressing protoplasts reached 1.8% as shown by an in situ β-glucuronidase assay. A duplicated 35S promoter with an alfalfa mosaic virus leader sequence (pBI-426) induced the highest expression rate among the constructs tested.

Embryogenic cell suspensions of cv. Bluggoe have also been bombarded with accelerated particles coated with a high expression uidA gene construct (pEmuGN) using a biolistic gun. After a partial optimization of the procedure, transient GUS assays reproducibly demonstrated the presence of 400 blue foci in 30 μl of settled cell volume (approximately 25 mg cells). Selection and characterization of antibiotic-resistant transformed cultures is in progress.

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Abbreviations

AMV:

alfalfa mosaic virus

GUS:

β-glucuronidase

TGE:

transient GUS expression

uidA :

gene for β-glucuronidase

References

  • Broekaert W.F., W. Marien, F.R.G. Terras, M.F.C.De Bolle, P. Proost, J.Van Damme, L. Dillen, M. Claeys, S.B. Rees, J. Vanderleyden & P.B.A. Cammue, 1992. Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins. Biochemistry 31: 4308–4314.

    Article  PubMed  CAS  Google Scholar 

  • Cammue B.P.A., M.F.C.De Bolle, F.R.G. Terras, P. Proost, J.Van Damme, S.B. Rees, J. Vanderleyden & W.F. Broekaert, 1992. Isolation and characterization of a novel class of plant antimicrobial peptides from Mirabilis jalapa L. seeds. J. Biol. Chem. 267: 2228–2233.

    PubMed  CAS  Google Scholar 

  • Cammue, B.P.A., M.F.C. De Bolle, F.R.G. Terras & W.F. Broekaert, 1993. Fungal disease control in Musa: application of new antifungal proteins. In: J. Ganry (Ed). Proc. Int. Symp. on Genetic Improvement of Bananas for Resistance to Diseases and Pests. Montpellier, France, September 7–9 1992, pp. 221–225. CIRAD/INIBAP/CTA.

  • Charest P.J., N. Caléro, D. Lachance, R.S.S. Datla, L.C. Duchesne & E.W.T. Tsang, 1993. Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the β-glucuronidase reporter gene. Plant Cell Rep. 12: 189–193.

    Article  CAS  Google Scholar 

  • Chibbar R.N., K.K. Kartha, R.S.S. Datla, N. Leung, K. Caswell, C.S. Mallard & L. Steinhauer, 1993. The effect of different promoter-sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells. Plant Cell Rep. 12: 506–509.

    Article  CAS  Google Scholar 

  • Chupeau M.-C., C. Bellini, P. Guerche, B. Maisonneuve, G. Vastra & Y. Chupeau, 1989. Transgenic plants of lettuce (Lactuca sativa) obtained through electroporation of protoplasts. Bio/Technology 7: 503–508.

    Article  Google Scholar 

  • Dhed'a, D., 1992. Culture de suspensions cellulaires embryogéniques et régénération en plantules par embryogénèse somatique chez le bananier et le bananier plantain (Musa spp.). Ph.D. thesis No. 1743. Faculty of Agricultural Sciences, Catholic University of Leuven, Belgium, 167 pp.

  • Dhed'a D., F. Dumortier, B. Panis, D. Vuylsteke & E.De Langhe, 1991. Plant regeneration in cell suspension cultures of the cooking banana cv. Bluggoe (Musa spp. ABB group). Fruits 46: 125–135.

    Google Scholar 

  • Dhir S.K., S. Dhir, A. Hepburn & J.M. Widholm, 1991. Factors affecting transient gene expression in electroporated Glycine max protoplasts. Plant Cell Rep. 10: 106–110.

    CAS  Google Scholar 

  • Diaz I. & P. Carbonero, 1992. Isolation of protoplasts from developing barley endosperm: a tool for transient expression studies. Plant Cell Rep. 10: 595–598.

    Article  Google Scholar 

  • FAO, 1993. Production yearbook 1992. Food and Agriculture Organisation of the United Nations, Rome, Italy, 265 pp.

    Google Scholar 

  • Finer J.J., P. Vain, M.W. Jones & M.D. McMullen, 1992. Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep. 11: 323–328.

    Article  CAS  Google Scholar 

  • Fromm M., L.P. Taylor & V. Walbot, 1985. Expression of genes transferred into monocot and dicot cells by electroporation. Proc. Natl. Acad. Sci. USA 82: 5824–5828.

    Article  PubMed  CAS  Google Scholar 

  • Hobbs S.L.A., J.A. Jackson, D.S. Baliski, C.M.O. DeLong & J.D. Mahon, 1990. Genotype- and promoter-induced variability in transient β-glucuronidase expression in pea protoplasts. Plant Cell Rep. 9: 17–20.

    Article  CAS  Google Scholar 

  • Huang Y.-W. & E.S. Dennis, 1989. Factors influencing stable transformation of maize protoplasts by electroporation. Plant Cell Tissue Organ Culture 18: 281–296.

    Article  CAS  Google Scholar 

  • Jefferson R.A., 1987. Assaying chimeric genes in plants: the GUS fusion system. Plant Molec. Biol. Rep. 5: 387–405.

    Article  CAS  Google Scholar 

  • Jobling S.A. & L. Gehrke, 1987. Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325: 622–625.

    Article  PubMed  CAS  Google Scholar 

  • Last D.I., R.I.S. Brettell, D.A. Chamberlain, A.M. Chaudhury, P.J. Larkin, E.L. Marsh, W.J. Peacock & E.S. Dennis, 1991. pEmu: an improved promoter for gene expression in cereal cells. Theor. Appl. Genet. 81: 581–588.

    Article  CAS  Google Scholar 

  • Lindsey K. & M.G.K. Jones, 1989. Stable transformation of sugarbeet protoplasts by electroporation. Plant Cell Rep. 8: 71–74.

    Article  Google Scholar 

  • Lis J.T., 1980. Fractionation of DNA fragments by polyethylene glycol induced precipitation. Meth. Enzymol. 65: 347–353.

    Article  PubMed  CAS  Google Scholar 

  • Megia R., R. Haicour, S. Tizroutine, V. Bui Trang, L. Rossignol, D. Sihachakr & J. Schwendiman, 1993. Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group). Plant Cell Rep. 13: 41–44.

    Article  CAS  Google Scholar 

  • Mobambo K., F. Gauhl, D. Vuylsteke, R. Ortiz, C. Pasberg-Gauhl & R. Swennen, 1993. Yield losses in plantain from black sigatoka leaf spot and field performance of resistant hybrids. Field Crops Res. 35: 35–42.

    Article  Google Scholar 

  • Murashige T. & R. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473–497.

    Article  CAS  Google Scholar 

  • Novak F.J., R. Afza, M.Van Duren, M. Perea-Dallos, B.V. Conger & T. Xiaolang, 1989. Somatic embryogenesis and plant regeneration in suspension cultures of dessert (AA and AAA) and cooking (ABB) bananas (Musa spp.). Bio/Technology 7: 154–159.

    Article  Google Scholar 

  • Panis B., D. Dehd'a & R. Swennen, 1992. Freeze-preservation of embryogenic Musa suspension cultures. In: R.P. Adams & J.E. Adams (Eds). Conservation of Plant Genes: DNA Banking and In Vitro Biotechnology, pp. 183–195. Academic Press, New York.

    Google Scholar 

  • Panis, B, D. Dhed'a, K. De Smet, L. Sagi, B. Cammue & R. Swennen, 1993a. Cell suspensions from somatic tissue in Musa: applications and prospects. In: J. Ganry (Ed). Proc. Int. Symp. on Genetic Improvement of Bananas for Resistance to Disease and Pests. Montpellier, France, September 7–9 1992, pp. 317–325. CIRAD/INIBAP/CTA.

  • Panis B., A.Van Wauwe & R. Swennen, 1993b. Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.). Plant Cell Rep. 12: 403–407.

    Article  Google Scholar 

  • Riggs C.D. & G.W. Bates, 1986. Stable transformation of tobacco by electroporation: Evidence for plasmid concatenation. Proc. Natl. Acad. Sci. USA 83: 5602–5606.

    Article  PubMed  CAS  Google Scholar 

  • Ritala A., L. Mannonen, K. Aspegren, M. Salmenkallio-Marttila, U. Kurtén, R. Hannus, J. Mendez Lozano, T.H. Teeri & V. Kauppinen, 1993. Stable transformation of barley tissue culture by particle bombardment. Plant Cell Rep. 12: 435–440.

    Article  CAS  Google Scholar 

  • Russell J.A., M.K. Roy & J.C. Sanford, 1992. Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation. Plant Physiol. 98: 1050–1056.

    Article  PubMed  CAS  Google Scholar 

  • Sagi L., S. Remy, B. Panis, R. Swennen & G. Volckaert, 1994. Transient gene expression in electroporated banana (Musa spp., cv. Bluggoe, ABB group) protoplasts isolated from regenerable embryogenic cell suspensions. Plant Cell Rep. 13: 262–266.

    Article  CAS  Google Scholar 

  • Sagi, L., B. Panis, S. Remy, H. Schoofs, K. De Smet, R. Swennen & B. Cammue, 1995. Genetic transformation of banana and plantain (Musa spp.) via practicle bombardment. Bio. Technology. in press.

  • Sanford J.C., F.D. Smith & J.A. Russell, 1993. Optimizing the biolistic process for different biological applications. Meth. Enzymol. 217: 483–509.

    Article  PubMed  CAS  Google Scholar 

  • Tada Y., M. Sakamoto & T. Fujimura, 1990. Efficient gene introduction into rice by electroporation and analysis of transgenic plants: use of electroporation buffer lacking chloride ions. Theor. Appl. Genet. 80: 475–480.

    Article  CAS  Google Scholar 

  • Takeuchi Y., M. Dotson & T. Keen, 1992. Plant transformation: a simple particle bombardment device based on flowing helium. Plant Molec. Biol. 18: 835–839.

    Article  CAS  Google Scholar 

  • Terras F.R.G., H.M.E. Schoofs, M.F.C.De Bolle, F.Van Leuven, S.B. Rees, J. Vanderleyden, B.P.A. Cammue & W.F. Broekaert, 1992. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J. Biol. Chem. 267: 15301–15309.

    PubMed  CAS  Google Scholar 

  • Toriyama K., Y. Arimoto, H. Uchimiya & K. Hinata, 1988. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology 6: 1072–1074.

    Article  CAS  Google Scholar 

  • Vain P., M.D.M. McMullen & J.J. Finer, 1993. Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep. 12: 84–88.

    Article  Google Scholar 

  • Vuylsteke D., R. Swennen & R. Ortiz, 1993. Development and early evaluation of black sigatoka-resistant tetraploid hybrids of plantain (Musa spp., ABB group). Euphytica 65: 33–42.

    Article  Google Scholar 

  • Wang Y.-C., T.M. Klein, M. Fromm, J. Cao, J.C. Sanford & R. Wu, 1988. Transient expression of foreign genes in rice, wheat and soybean cells following particle bombardment. Plant Molec. Biol. 11: 433–439.

    Article  CAS  Google Scholar 

  • Zhang W. & R. Wu, 1988. Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants. Theor. Appl. Genet. 76: 835–840.

    Article  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Tropical Crop Husbandry, Catholic University of Leuven, Belgium

    László Sági, Serge Remy, Bert Verelst, Bart Panis & Rony Swennen

  2. F.A. Janssens Laboratory of Genetics, Catholic University of Leuven, Belgium

    Bruno P. A. Cammue

  3. Laboratory of Gene Technology, Catholic University of Leuven, Belgium

    Guido Volckaert

Authors
  1. László Sági
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  2. Serge Remy
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  3. Bert Verelst
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  4. Bart Panis
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  5. Bruno P. A. Cammue
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  6. Guido Volckaert
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  7. Rony Swennen
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Sági, L., Remy, S., Verelst, B. et al. Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions. Euphytica 85, 89–95 (1995). https://doi.org/10.1007/BF00023934

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