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Stimulation of growth of culturedNicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts

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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 μM 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 μM due to chelation by amino-acids, and similar tube morphologies were obtained with 1.0–1.5 μM added CuSO4 when NH4Cl replaced the amino-acids. When the medium containing amino-acids was supplemented with both 12.5% PEG-6000 and 30 μM 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 μm/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 μM) had a similar morphogenetic effect to CuSO4, but addition of 300 μM 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.

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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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  1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3052, Parkville, Vic., Australia

    S. M. Read, A. E. Clarke & A. Bacic

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  1. S. M. Read
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  2. A. E. Clarke
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  3. A. Bacic
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Read, S.M., Clarke, A.E. & Bacic, A. Stimulation of growth of culturedNicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts. Protoplasma 177, 1–14 (1993). https://doi.org/10.1007/BF01403393

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