Stimulation of growth of culturedNicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts |
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Authors: | S M Read A E Clarke A Bacic |
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Institution: | (1) Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3052 Parkville, Vic., Australia |
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Abstract: | Summary Growth of pollen tubes ofNicotiana tabacum W 38 in a defined liquid medium buffered at pH 5.9 and containing sucrose, amino-acids, boric acid, salts and an antibacterial agent was stimulated by the addition of poly(ethylene glycol) 6000 (PEG-6000) and Cu(II) salts. In the absence of both these supplements, up to 50% of the hydrated pollen grains did not develop further, and the germinated tubes were slow-growing and abnormal, with thickened walls, kinked growth, and fragile, swollen tips containing granular cytoplasm. Addition of 10–15% (w/v) purified PEG-6000 increased germination to 80–90% and prevented the progressive bursting of pollen grains and tube tips, but growth was still slow and kinked and tips remained swollen. Addition of 30 M CuSO4 did not stimulate germination or prevent tip bursting, but produced straight-growing tubes with smooth-sided tips resembling the tips of tubes growing through stylar tissue; the free Cu2+ concentration under these conditions was about 1.0 M due to chelation by amino-acids, and similar tube morphologies were obtained with 1.0–1.5 M added CuSO4 when NH4Cl replaced the amino-acids. When the medium containing amino-acids was supplemented with both 12.5% PEG-6000 and 30 M CuSO4, long-term (48 h) growth of straight pollen tubes with smooth-sided tips, thin walls and long ladders of callose plugs was observed; growth occurred at 250 m/h, approximately 30–40% of the rate observed in the style. Although omission of CuSO4 from this complete medium severely affected tube growth and callose plug deposition, it did not alter the timing of generative-nucleus division, and thus the different parameters associated with the second phase of pollen-tube growth can be uncoupled in culture. High levels of FeSO4 (300 M) had a similar morphogenetic effect to CuSO4, but addition of 300 M L-ascorbate or D-iso-ascorbate was required to prevent precipitation of Fe(III) oxide and prolong the stimulation of pollen-tube growth; EDTA removed the morphogenetic effect of both CuSO4 and FeSO4. Further, an impure grade of PEG-4000 was contaminated with an organic morphogen that allowed continued slow growth of pollen tubes with smooth, straight-sided tips in the absence of added CuSO4 or FeSO4, with tube morphology unaffected by ascorbate or EDTA. However, the long-term morphogenetic effect of trace levels of CuSO4 suggests that Cu(II) salts play an important role in pollen-tube development in at least this species ofNicotiana.Abbreviations A475
absorbance at 475 nm
- DAPI
4 ,6-diamidino-2-phenylindole
- EDTA
ethylene-diamine N,N,N ,N -tetraacetic acid
- MES
2-(N-morpholino)-ethane sulphonic acid
- OG
ordinary grade of poly(ethylene glycol)
- PEG
poly(ethylene glycol)
- SP
Specially Purified for Biochemistry grade of poly(ethylene glycol) |
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Keywords: | Copper Nicotiana Pollen Pollen tube Poly(ethylene glycol) Tip growth |
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