Stable transformation of a recalcitrant kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues



An efficient method to produce highly regenerative tissues from seeds of a previously recalcitrant cultivar of Kentucky bluegrass (Poa pratensis L. ev. Kenblue) was established under dim-light conditions (10–30 μE m−2s−1, 16-h light) using media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5 or 9.0 μM), 6-benzylaminopurine (BA; 0.44 or 2.2 μM), and a high level of cupric sulfate (5.0 μM). The tissues were co-transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA; gus), and a synthetic green fluorescent protein gene [sgfp(S65T)]. From 463 individual explants bombarded, 10 independent transgenic events (2.2%) were obtained after a 3–4-month selection period for hygromycin resistance using 30–100 mg l−1 hygromycin B; of the 10 independent events, seven (70%) were regenerable. Stable integration of the transgene(s) in transgenic plants was confirmed by polymerase chain reaction and DNA blot hybridization analyses. Coexpression frequency of all three genes was 20%; for two transgenes, either hpt/uidA or hpt/sgfp(S65T), coexpression frequency was 30–40%.

Key words

Poa pratensisKenblue recalcitrant cultivar transformation highly regenerative tissue transgene expression 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bashaw, E. C.; Funk, R. C. Apomictic grasses. In: Fehr, W. R., ed. Principles of cultivar development. Vol. 2. New York: Macmillan Publishing; 1987:40–82.Google Scholar
  2. Boyd, L. A.; Dale, P. J. Callus production and plant regeneration from mature embryos of Poa pratensis L. Plant Breeding 97:246–254; 1986.CrossRefGoogle Scholar
  3. Chiu, W.-L.; Niwa, Y.; Zeng, W.; Hirano, T.; Kobayashi, H.; Sheen, J. Engineered GFP as a vital reporter in plants. Curr. Biol. 6:325–330; 1996.PubMedCrossRefGoogle Scholar
  4. Cho, M.-J.; Ha, C. D.; Lemaux, P. G. Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues. Plant Cell Rep. 19:1084–1089; 2000.CrossRefGoogle Scholar
  5. Cho, M.-J.; Jiang, W.; Lemaux, P. G. Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci. 138:229–244; 1998.CrossRefGoogle Scholar
  6. Cho, M.-J.; Jiang, W.; Lemaux, P. G. High-frequency transformation of oat via microprojectile bombardment of seed-derived highly regenerative cultures. Plant Sci. 148:9–17; 1999.CrossRefGoogle Scholar
  7. Christensen, A. H.; Quail, P. H. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5:1–6; 1996.CrossRefGoogle Scholar
  8. Dellaporta, S. Plant DNA miniprep and microprep. In: Freeling, M.; Walbot, V., eds. The maize handbook. New York: Springer-Verlag; 1994:522–525.Google Scholar
  9. Duell, R. W. The blucgrasses. In: Barnes, R. F.; Metcalfe, D. S.; Heath, M. E., eds. Forages: the science of grassland agriculture. Ames: Iowa State University Press; 1985:188–197.Google Scholar
  10. Etter, A. G. How Kentucky bluegrass grows. Ann. Missouri Bot. Garden 38:293–367; 1951.CrossRefGoogle Scholar
  11. Feinberg, A. P.; Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6–13; 1982.CrossRefGoogle Scholar
  12. Germplasma Resources Information (GRIN) USDA/ARS Germplasm Services Laboratory. Beltsville MD: Database Management Unit; 1991.Google Scholar
  13. Griffin, J. D.; Dibble, M. S. High-frequency plant regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.). Plant Cell Rep. 14:721–724; 1995.CrossRefGoogle Scholar
  14. Hodges, C. F.; Stephens, L. C.; Campbell, D. A. Ethylene and ethane from Poa pratensis callus and from leaf blades of regenerated and seed-derived plants inoculated with Bipolaris sorokiniana. J. Plant Physiol. 154:113–118; 1999.Google Scholar
  15. Hunter, C. P. Plant regeneration from microspores of barley. Hordeum rulgare. PhD thesis. Ashford, Kent: Wye College, University of London; 1988.Google Scholar
  16. Jefferson, R. A.; Kavanagh, T. A.; Bevan, M. W. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901–3907; 1987.PubMedGoogle Scholar
  17. Kasha, K. J.; Ziauddin, A.; Cho, U.-H. Haploids in creal improvement anther and microspore culture. In: Gustafson, J. P., ed. Gene manipulation in plant improvement, II; 19th Stadler Genetics Symposium, New York: Plenum Press; 1990:213–236.Google Scholar
  18. Ke, S.; Lee, C. W. Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures. Plant Cell Rep. 15:882–887; 1996.CrossRefGoogle Scholar
  19. Ke, S.; Lee, C. W.; Cheng, Z.-M. Genetic transformation of Kentucky bluegrass with rolC gene. Lexington, Kentucky, USA: 93rd Annual Conference of American Society for Horticultural Science. Hortscience 31:616 (abstr.); 1996.Google Scholar
  20. Kim, H.-K.; Lemaux, P. G.; Buchanan, B. B.; Cho, M.-J. Reduction of genotype limitation in wheat (Triticum aestivum L.) transformation. Congress on In Vitro Biol., New Orleans, LA. 5–9th June. In Vitro Cell. Dev. Biol. Plant 35 (3 II); 1021 (abstr.); 1999.Google Scholar
  21. Krans, J. V. Cell culture of turfgrasses. In: Sheard, R. W., ed. Proc. 4th Int. Turfgrass Res. Conference, University of Guelph, Ontario, Canada, July 19–23, 1981. Guelph, Ontario: University of Guelph Press; 1981:27–33.Google Scholar
  22. Lemaux, P. G.; Cho, M.-J.; Louwerse, J.; Williams, R.; Wan, Y. Bombardment-mediated transformation methods for barley. Bio-Rad Bulletin 2007:1–6; 1996.Google Scholar
  23. Lemaux, P. G.; Cho, M.-J.; Zhang, S.; Bregitzer, P. Transgenic cereals: Hordeum vulgare (barley). In: Vasil, I. K., ed. Molecular improvement of cereal crops. London: Kluwer Academic Publishers; 1999:225–316.Google Scholar
  24. Manton, M.; Riordan, T. P.; Shearman, R. C. Callus induction and plant regeneration in Kentucky bluegrass (Poa pratensis L.). Proc. Nebraska Acad. Sci. 80:1982.Google Scholar
  25. McDonnell, R. E.; Conger, B. V. Callus induction and plantlet formation from mature embryo explants of Kentucky bluegrass. Crop Sci. 24:573–578; 1984.CrossRefGoogle Scholar
  26. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.CrossRefGoogle Scholar
  27. Nielsen, K. A.; Knudsen, E. regeneration of green plants from embryogenic suspension cultures of Kentucky bluegrass (Poa pratensis L.). J. Plant Physiol. 141:589–595; 1993.Google Scholar
  28. Nielsen, K. A.; Larsen, E.; Knudsen, E. Regeneration of protoplast-derived green plants of Kentucky bluegrass (Poa pratensis L.). Plant Cell Rep. 12:537–540; 1993.CrossRefGoogle Scholar
  29. Olsen, F. L. Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare. The effects of ammonium nitrate, glutamine and asparagine as nitrogen sources. Carlsberg Res. Comm. 52:393–404; 1987.CrossRefGoogle Scholar
  30. Sharman, B. C. The biology and developmental morphology of the shoot apex in the Gramineae. New Phytol. 46:20–34; 1947.CrossRefGoogle Scholar
  31. Sørensen, M. B.; Müller, M.; Skerritt, J.; Potrykus, I. Hordein promoter methylation and transcriptional activity in wild-type and mutant barley endosperm. Mol. Gen. Genet. 250:750–760; 1996.PubMedCrossRefGoogle Scholar
  32. Sticklen, M. B.; Kenna, M. P. Turfgrass biotechnology; cell and molecular genetic approaches to turfgrass improvement. Chelsea, MI: Ann Arbor Press; 1998.Google Scholar
  33. Stubbs, L.; Huxley, C.; Hogan, B.; Evans, T.; Fred, M.; Duboule, D.; Lehrach, H. The hox-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2. Genomics 6:645–650; 1990.PubMedCrossRefGoogle Scholar
  34. Van Ark, H. F.; Zaal, M. A. C. M.; Creemers-Molenaar, J.; Van der Valk, P. Improvement of the tissue culture response of seed-derived callus cultures of Poa pratensis L.: effect of gelling agent and abscisic acid. Plant Cell Tiss. Organ Cult. 27:275–280; 1991.CrossRefGoogle Scholar
  35. Van der Valk, P.; Ruis, F.; Tettelaar-Schrier, A. M.; Van de Velde, C. M. Optimizing plant regeneration from seed-derived callus cultures of Kentucky bluegrass. The effect of benzyladenine. Plant Cell Tiss. Organ Cult. 40:101–103; 1995.CrossRefGoogle Scholar
  36. Van der Valk, P.; Zaal, M. A. C. M.; Creemers-Molenaar, J. Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. Plant Cell Rep. 7:644–647; 1989.Google Scholar
  37. Wan, Y.; Lemaux, P. G. Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104:37–48; 1994.PubMedGoogle Scholar
  38. Wu, L.; Jampates, R. Chromosome number and isoenzyme variation in Kentucky bluegrass cultures and plants regenerated from tissue culture. Cytologia 51:125–132; 1986.Google Scholar
  39. Zhang, S.; Cho, M.-J.; Bregitzer, P.; Lemaux, P. G. Comparative analysis of genomic DNA methylation status and field performance of plants derived from embryogenic calli and shoot meristematic cultures. In: Altman, A.; Ziv, M.; Izhar, S., eds. Plant biotechnology and in vitro biology in the 21st century. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1999:263–267.Google Scholar

Copyright information

© Society for In Vitro Biology 2001

Authors and Affiliations

  1. 1.Department of Plant and Microbial BiologyUniversity of CaliforniaBerkeleyUSA

Personalised recommendations