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Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

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Summary

The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.

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References

  • Altpeter, F.; Vasil, V.; Srivastava, V.; Stoger, E.; Vasil, I. K. Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Rep. 16:12–17; 1996.

    CAS  Google Scholar 

  • Bizily, S. P.; Rugh, C. L.; Summers, A. O.; Meagher, R. B. Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials. Proc. Natl Acad. Sci. USA 96:6808–6813; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Bower, R.; Elliott, A. R.; Potier, B. A. M.; Birch, R. G. High-efficiency, microprojectile-mediated cotransformation of sugarcane using visible or selectable markers. Mol. Breed. 2:239–249; 1996.

    Article  CAS  Google Scholar 

  • Carlson, A. R.; Letarte, J.; Chen, J.; Kasha, K. J. Visual selection of microspore-derived transgenic barley (Hordeum vulgare L) with green fluorescent protein. Plant Cell Rep. 20:331–337; 2001.

    Article  CAS  Google Scholar 

  • Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263:802–805; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Cheng, M.; Jarret, R. L.; Li, Z.; Demski, J. W. Production of fertile transgenic peanut (Archis hypogaea L.) plants using Agrobacterium tumefaciens. Plant Cell Rep. 15:653–657; 1996.

    Article  CAS  Google Scholar 

  • Cho, H. J.; Widholm, J. M. Agrobacterium tumefacients-mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression. Plant Cell Tiss. Organ Cult. 69:251–258; 2002.

    Article  CAS  Google Scholar 

  • Choi, H. W.; Lemaux, P. G.; Cho, M.-J. Selection and osmotic treatment exacerbate cytological aberrations in transformed barley (Hordeum vulgare) J. Plant Physiol. 158:935–943; 2001.

    Article  CAS  Google Scholar 

  • Datla, R. S. S.; Bekkaoui, F.; Hammerlindl, J. K.; Pilate, G.; Dunstan, D. I.; Crosby, W. L. Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci. 94:139–149; 1993.

    Article  CAS  Google Scholar 

  • de Ruijter, N. C. A.; Verhees, J.; van Leeuwen, w.; van der Krol, A. R. Evaluation and comparison of the GUS, LUC; and GFP reporter system for gene expression studies in plants. Plant Biol. 5:103–115; 2003.

    Article  Google Scholar 

  • Elliott, R.; Campbell, J. A.; Dugdale, B.; Brettell, R. I. S.; Grof, C. P. L. Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Rep. 18:707–714; 1999.

    Article  CAS  Google Scholar 

  • Fleming, G. H.; Olivares-Fuster, O.; Fatta Del-Bosso, S.; Grosser, J. W. An alternative method for the genetic transformation of sweet orange. In Vitro Cell. Dev. Biol. Plant 36:450–455; 2000.

    Article  CAS  Google Scholar 

  • Garbarino, J. E.; Belknap, W. R. Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol. Biol. 24:119–127; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Hansch, R.; Koprek, T.; Mendel, R. R.; Schulze, J. An improved protocol for eliminating endogenous beta-glucuronidase background in barley. Plant Sci. 105:63–69; 1995.

    Article  Google Scholar 

  • Harper, B. K.; Mabon, S. A.; Leffel, S. M.; Halfhill, M. D.; Richards, H. A.; Moyer, K. A.; Stewart, C. N. Jr. Green fluorescent protein as a marker for expression of a second gene in transgenic plants. Nat. Biotechnol. 17:1125–1129; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Hodal, L.; Bochardt, A.; Nielsen, J. E.; Mattsson, O. O.; Finn, T. Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and nontransgenic plants. Plant Sci. 87:115–122; 1992.

    Article  CAS  Google Scholar 

  • Huber, M.; Hahn, R.; Hess, D. High transformation frequencies obtained from a commercial wheat (Triticum aestivum L. cv. ‘Combi’) by microbombardment of immature embryos followed by GFP screening combined with PPT selection. Mol. Breed. 10:19–30; 2002.

    Article  CAS  Google Scholar 

  • Iglesias, V. A.; Gisel, A.; Bilang, R.; Leduc, N.; Potrykus, I.; Sautter, C. Transient expression of visible marker genes in meristem cells of wheat embryos after ballistic micro-targeting. Planta 192:84–91; 1994.

    CAS  Google Scholar 

  • Jordan, M. C. Green fluorescent protein as a visual marker for wheat transformation. Plant Cell Rep. 19:1069–1075; 2000.

    Article  CAS  Google Scholar 

  • Kaeppler, H. F.; Carlson, A. R.; Menon, G. K. Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation. In Vitro Cell. Dev. Biol. Plant 37:120–126; 2001.

    Article  CAS  Google Scholar 

  • Kaeppler, H. F.; Menon, G. K.; Skadsen, R. W.; Nuutila, A. M.; Carlson, A. R. Transgenic oat plants via visual selection of cells expressing green fluorescent protein. Plant Cell Rep. 19:661–666; 2000.

    Article  CAS  Google Scholar 

  • Kemper, E. L.; Marcio, J. L.; Paulo, A. Effect of microprojectile bombardment parameters and osmotic freatments on particle penetration and tissue damage in transiently transformed cultured immature maize (Zea mays L.) embryos. Plant Sci. 121:85–93; 1996.

    Article  Google Scholar 

  • Knauft, D. A.; Ozias-Akins, P. Recent methodologies for germplasm enhancement and breeding. In: Pattee, H. E.; Stalker, H. T., eds. Stillwater, OK: American Peanut. Research and Education Society; 1995:54–94.

    Google Scholar 

  • Leffel, S. M.; Mabon, S. A.; Stewart, C. N. Jr Applications of green fluorescent protein in plants. Biotechniques 23:912–918; 1997.

    PubMed  CAS  Google Scholar 

  • Livingstone, D. M.; Birch, R. G. Plant regeneration and microprojectilemediated gene transfer in embryonic leaflets of peanut (Arachis hypogaea L.). Aust. J. Plant Physiol. 22:585–591; 1995.

    Article  CAS  Google Scholar 

  • Livingstone, D. M.; Birch, R. G. Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaca L.) by particle bombardment into embryogenic callus produced from mature seeds. Mol. Breed. 5:43–51; 1999.

    Article  Google Scholar 

  • Ludwig, S. R.; Bowen, B.; Beach, L.; Wessler, S. R. A regulatory gene as a novel visible marker for maize transformation. Science 247:449–450; 1990.

    Article  PubMed  Google Scholar 

  • McKently, A. H.; Moore, G. A.; Gardner, F. P. Direct somatic embryogenesis from axes of mature peanut embryos. In Vitro Cell. Dev. Biol. Plant 27:197–200; 1991.

    Google Scholar 

  • Molinier, J.; Himber, C.; Hahne, G. Use of green fluorescent protein for detection of transformed shoots and homozygous offspring. Plant Cell Rep. 19:219–223; 2000.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Murray, M. G.; Thompson, W. F. Rapid isolation of high molecular weight plant DNA. Nucl. Acids Res. 8:4321–4325; 1980.

    Article  PubMed  CAS  Google Scholar 

  • Nageshwara Rao, R. C.; Nigam, S. N. Genetic options for drought management in groundnut. In: Saxena, N. P., ed. Management of agricultural drought—agronomic and genetic options. Enfield, NH: Science Publishers, Inc; 2003:123–141.

    Google Scholar 

  • Niedz, R. P.; Sussman, M. R.; Satterlee, J. S. Green fluorescent protein: an in vivo reporter of plant gene expression. Plant Cell Rep. 14:403–406; 1995.

    Article  CAS  Google Scholar 

  • Ozias-Akins, P.; Gill, R. Progress in the development of tissue culture and transformation methods applicable to the production of transgenic peanut. Peanut Sci. 28:123–131; 2001.

    Article  CAS  Google Scholar 

  • Ozias-Akins, P.; Schnall, J. A.; Anderson, W. F.; Singsit, C.; Clemente, T. E.; Adang, M. J.; Weissinger, A. K. Regeneration of transgenic peanut plants from stably transformed embryogenic callus. Plant Sci. 93:185–194; 1993.

    Article  CAS  Google Scholar 

  • Perl, A.; Kless, H.; Blumenthal, A.; Galili, G.; Galun, E. Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization. Mol. Gen. Genet. 235:279–284; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Rasco-Gaunt, S.; Riley, A.; Barcelo, P.; Lazzeri, P. A. Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues. Plant Cell Rep. 19:118–127; 1999.

    Article  CAS  Google Scholar 

  • Remans, T.; Schenk, P. M.; Manners, J. M.; Grof, C. P. L.; Elliott, A. R. A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants. Plant Mol. Biol. Rep. 17:385–395; 1999.

    Article  CAS  Google Scholar 

  • Richards, H. A.; Halfhill, M. D.; Millwood, R. J.; Stewart, C. N. Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants. Plant Cell Rep. 22:117–121; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Samoylov, V. M.; Tucker, D. M.; Parrott, W. A. Soybean [Glycine max (L.) Merrill] embryogenic cultures: the role of sucrose and total nitrogen content on proliferation. In Vitro Cell. Dev. Biol. Plant 34:8–13; 1998.

    CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Schöpke, C.; Taylor, N. J.; Cárcamo, R.; Beachy, R. N.; Fauquet, C. Optimization of parameters for particle bombardment of embryogenic suspension cultures of cassava (Manihot esculenta Crantz) using computer image analysis. Plant Cell Rep. 16:526–530; 1997.

    Article  Google Scholar 

  • Sharma, H. C.; Sharma, K. K.; Seetharama, N.; Ortiz, R. Genetic transformation of crop plants: risks and opportunities for the rural poor. Curr. Sci. 80:1495–1503; 2001.

    CAS  Google Scholar 

  • Singsit, C.; Adang, M. J.; Lynch, R. E.; Anderson, W. F.; Wang, A. M.; Cardinean, G.; Ozias-Akins, P. Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer. Transgenic Res. 6:169–176; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Stalker, H. T.; Simpson, C. E. Germplasm resources in Arachis. In: Pattee, H. E.; Stalker, H. T., eds. Stillwater, OK: American Peanut Research and Education Society; 1995:14–53.

    Google Scholar 

  • Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67:509–544; 1998.

    Article  PubMed  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Wang, A. M.; Fan, H. L.; Singsit, C.; Ozias-Akins, P. Transformation of peanut with a soybean vspB promoter-uidA chimeric gene I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants. Plysiol. Plant. 102:38–48; 1998.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Horticulture, University of Georgia, Tifton Campus, 31793, Tifton, GA

    Madhumita Joshi, Chen Niu, Geraldine Fleming, Sulekha Hazra, Ye Chu, Hongyu Yang & Peggy Ozias-Akins

  2. School of Forest Resources, University of Georgia, 30602, Athens, GA

    C. Joseph Nairn

Authors
  1. Madhumita Joshi
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  2. Chen Niu
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  3. Geraldine Fleming
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  4. Sulekha Hazra
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  5. Ye Chu
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  6. C. Joseph Nairn
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  7. Hongyu Yang
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  8. Peggy Ozias-Akins
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Correspondence to Peggy Ozias-Akins.

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Joshi, M., Niu, C., Fleming, G. et al. Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation. In Vitro Cell.Dev.Biol.-Plant 41, 437–445 (2005). https://doi.org/10.1079/IVP2005676

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