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Preparation of Sporoplasts for Studies of Pollen Physiology

  • Bruce G. Baldi
  • John D. Everard
  • Frank A. Loewus
Conference paper

Abstract

Sporoplasts (pollen protoplasts) are produced in quantity from pollen of Lilium longiflorum Thunb. by exposure to 4-methylmorpholine N-oxide (MMNO). Treatment for 1 h at 25°C with aqueous MMNO, 15% w/v, in buffered solution, pH 5.3, containing 0.5% cellulase, 0.25% pectinase and 0.1% bovine serum albumin loosens the attachment of the sporoplast to exine but fails to effect release. Displacement from exine is achieved by a series of three treatments. First, MMNO-treated pollen is suspended in a buffer containing 0.5 mM EDTA, 0.6 M mannitol and 10 mM MES, pH 5.5 which effects release of sporoplasts from exines (25–30%). Then it is suspended in a buffer containing 5 mM CaCl2, 0.6 M mannitol and 5 roM MES, pH 5.5. Finally, it is suspended in 5 mM CaCl2, 0.3 M pentaerythritol and 3 mM MES, pH 5.3. Separation of sporoplasts from exines and undigested pollen is accomplished on a discontinuous sucrose density gradient. Purified sporoplasts are readily ruptured by passing the suspension through a 15 um3 nylon mesh. Data is presented on cell-free distribution within selfgenerated Percoll gradients of phytase and a phytate-rich component.

Keywords

Pollen Tube Phytic Acid Mature Pollen Phytase Activity Percoll Gradient 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Reference

  1. Baldi, B.G., V.R. Franceschi and F.A. Loewus, 1986. Dissolution of pollen intine and release of sporoplasts. Mulcahy, G, Mulcahy Bergamini and E. Ottaviano, eds., Biotechnology and ecology of pollen. Springer, New York. pp 77–82.CrossRefGoogle Scholar
  2. Baldi, B.G., V.R. Franceschi and F.A. Loewus 1987a. Localization of phosphorus and cation reserves in Lilium longiflorum pollen. Plant Physiol. 85:1018–1021.CrossRefGoogle Scholar
  3. Baldi, B.G., V.R. Franceschi and F.A. Loewus 1987b. Preparation and properties of pollen sporoplasts. Protoplasma 141:47–55.CrossRefGoogle Scholar
  4. Baldi, B.G., V.R. Franceschi, F.A. Loewus, J.J. Scott, J.D. Everard and F.A. Loewus 1988. Localization of constitutive phytases in lily pollen and properties of the pH 8 form. Plant Sci. (in press).Google Scholar
  5. Bajaj, Y.P.S. 1974. Isolation and culture studies on pollen tetrad and pollen mother cell protoplasts. Plant Sci. Lett. 3:93–99.CrossRefGoogle Scholar
  6. Bhojwani, S.S. and E.C. Cocking 1972. Isolation of protoplasts from pollen tetrads. Nature New BioI. 239:29–30.CrossRefGoogle Scholar
  7. Espelie, K., F.A. Loewus, R.J. Pugmire, B.G. Baldi, and P.H. Given. Structural studies of Lilium longiflorum sporopollenin by 13C-NMR. (submitted for publication).Google Scholar
  8. Given, P. H., N. J. Ryan-Gray, G. Davidonis, P. C. Painter and A. Traverse 1988. The chemical structure of sporopollenin, the precursor of sporinite macerals. Org. Geochem. in press.Google Scholar
  9. Imamura, J. and I. Potrykus 1983. Isolated tetrad protoplasts develop to the binucleate stage in tobacco (Nicotiana tabacum cv. Havana) Sixth Inter. Protoplast Symposium, poster proceedings, Protoplasts 1983, pp 15.Google Scholar
  10. Ito, M. 1973. Studies on the behavior of meiotic protoplasts I. Isolation from microsporocytes of Liliaceous plants. Bot. Mag. Tokyo 86:133–141.CrossRefGoogle Scholar
  11. Latta, M. and M. Eskin, 1980. A simple and rapid colorimetric method for phytate determination. J. Agric. Food Chem. 28:1313–1317.CrossRefGoogle Scholar
  12. Loewus, F.A., B.G. Baldi, V.R. Franceschi, L.D. Meinert and J.J. McCollum, 1985. Pollen sporoplasts: Dissolution of pollen walls. Plant Physiol. 78:652–654.PubMedCrossRefGoogle Scholar
  13. Maeda, M., M. Yoshioka and M. Ito, 1979. Studies on the behavior of meiotic protoplasts IV. Protoplasts isolated from microsporocytes of Liliaceous plants. Bot. Mag. Tokyo 92:111–121.CrossRefGoogle Scholar
  14. Nunes, S.P. and F. Galemback 1985. Percoll and Ficoll selfgenerated density gradients by low speed osmocentrifugation. Anal Biochem. 146:48–51.PubMedCrossRefGoogle Scholar
  15. Power, J.B., 1973. Isolation of mature tobacco pollen protoplasts. in: H.E. Street, ed., Plant tissue and cell culture. Blackwell Sci. Publ., Oxford. pp 118–119.Google Scholar
  16. Rajasekhar, E.W., 1973. Nuclear divisions in protoplasts isolated from pollen tetrads of Datura metel. Nature 246:223–224.CrossRefGoogle Scholar
  17. Scott, J.J. and F.A. Loewus, 1986a. Phytate metabolism in plants. in: E. Graf, ed., Phytic Acid: Chemistry and Applications. Pilatus Press, Minneapolis. pp23–42.Google Scholar
  18. Scott, J.J. and F.A. Loewus, 1986b. A calcium-activated phytase from pollen of Lilium longiflorum. Plant Physiol. 82:333–335.PubMedCrossRefGoogle Scholar
  19. Tanaka, I., C. Kitazume and M. Ito, 1987. The isolation and culture of lily pollen protoplasts. Plant Sci. 50:205–211.CrossRefGoogle Scholar
  20. Zhu, C., Y. Xie and S. Hu, 1983. Isolation and cultural behavior of pollen tube subprotoplasts in Antirrhinum majus L. Acta Bot. sinica 26:459–465.Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1988

Authors and Affiliations

  • Bruce G. Baldi
    • 1
  • John D. Everard
    • 1
  • Frank A. Loewus
    • 1
  1. 1.Institute of Biological ChemistryWashington State UniversityPullmanUSA

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