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Effect of n-butanol and cold pretreatment on the cytoskeleton and the ultrastructure of maize microspores when cultured in vitro

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Abstract

The improvement of androgenic induction efficiency of anther cultures is an important goal for plant biotechnology. Although n-butanol has been proven to enhance the androgenic induction, the structural background of this effect has not been investigated in detail. In the present study, the cytological and ultrastructural alterations triggered by two treatments that improve androgenic induction, n-butanol (0.2 % n-butanol for 6 h) and cold pretreatment (7 °C for 10 days) were studied in maize anther cultures. Both treatments increased the frequency of responding microspores, and the highest embryo yield (20.9 embryos per 100 plated anthers compared to 0.5/100 anthers in control) was achieved when a combination of both treatments was applied. To study the effect of the treatments on the cytoskeleton, we labeled microtubules using indirect immunofluorescence and actin filaments by rhodamine phalloidin. Cold pretreatment increased the quantity of actin filaments, whereas the microtubule network remained unaffected. In contrast, n-butanol treatment triggered the reversible depolymerization of microtubules, without having any effect on the actin network. Transmission electron microscopy revealed that n-butanol induced the formation of irregular cell walls. Autophagy-related structures were present during the early development of embryogenic microspores following both treatments, but autophagy was only sustained after fourteen days in microspore-derived structures treated with n-butanol. The results support the concept that the androgenic developmental switch is assisted by cytoskeletal rearrangements, which may facilitate androgenic induction through the promotion of symmetric divisions. The longer duration of autophagic processes may also play a role in the elevated embryo induction after n-butanol treatment.

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Acknowledgments

This work was supported by Hungarian Scientific Research Fund grant No. OTKA 80260 and the GENPROF IF-18/2012 Research Infrastructure Grant from the Hungarian Academy of Sciences. The authors wish to thank Victor Žárský, Lukas Synek and Roman Pleskot for kindly sharing methods for the fluorescent visualisation of the plant cytoskeleton, and Erika Gondos, Emese Bék and Szilvia Fodor for their excellent technical assistance.

Author contributions

AF planned and performed the cytological and ultrastructural analysis, carried out the statistical analysis, interpreted the results and drafted the manuscript. BB conceived the study aims, and critically reviewed the manuscript; KJ contributed to the drafting and critically reviewed the manuscript. PKFF and HA performed and interpreted the results of the anther culture experiments, LSZ contributed to the ultrastructural analysis and critically reviewed the manuscript. All authors approved the final version of the article.

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  1. Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Martonvásár, Hungary

    A. Fábián, P. K. Földesiné Füredi, H. Ambrus, K. Jäger & B. Barnabás

  2. Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary

    L. Szabó

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  1. A. Fábián
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Correspondence to K. Jäger.

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Fábián, A., Földesiné Füredi, P.K., Ambrus, H. et al. Effect of n-butanol and cold pretreatment on the cytoskeleton and the ultrastructure of maize microspores when cultured in vitro. Plant Cell Tiss Organ Cult 123, 257–271 (2015). https://doi.org/10.1007/s11240-015-0829-9

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