Abstract

BackgroundA mannitol stress treatment and a subsequent application of n-butanol, known as a microtubule-disrupting agent, enhance microspore embryogenesis (ME) induction and plant regeneration in bread wheat. To characterize changes in cortical (CMT) and endoplasmic (EMT) microtubules organization and dynamics, associated with ME induction treatments, immunocytochemistry studies complemented by confocal laser scanning microscopy (CLSM) were accomplished. This technique has allowed us to perform advanced 3- and 4D studies of MT architecture. The degree of MT fragmentation was examined by the relative fluorescence intensity quantification.ResultsIn uni-nucleated mannitol-treated microspores, severe CMT and EMT fragmentation occurs, although a complex network of short EMT bundles protected the nucleus. Additional treatment with n-butanol resulted in further depolymerization of both CMT and EMT, simultaneously with the formation of MT aggregates in the perinuclear region. Some aggregates resembled a preprophase band. In addition, a portion of the microspores progressed to the first mitotic division during the treatments. Bi-nucleate pollen-like structures showed a high MT depolymerization after mannitol treatment and numerous EMT bundles around the vegetative and generative nuclei after n-butanol. Interestingly, bi-nucleate symmetric structures showed prominent stabilization of EMT.ConclusionsFragmentation and stabilization of microtubules induced by mannitol- and n-butanol lead to new configurations essential for the induction of microspore embryogenesis in bread wheat. These results provide robust insight into MT dynamics during EM induction and open avenues to address newly targeted treatments to induce ME in recalcitrant species.

Highlights

  • A mannitol stress treatment and a subsequent application of n-butanol, known as a microtubuledisrupting agent, enhance microspore embryogenesis (ME) induction and plant regeneration in bread wheat

  • Cultures of bread wheat isolated microspores used in this study were characterized morphologically and by their Doubled haploid (DH) plant production efficiency to verify the effect of a mannitol or a mannitol plus n-butanol treatment

  • After treatment with 0.7 M mannitol for five days at 25°C (MAN), most microspores showed a vacuole partially fragmented by cytoplasmic strands and the nucleus located on the opposite side of the operculum (Fig. 1b)

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Summary

Introduction

A mannitol stress treatment and a subsequent application of n-butanol, known as a microtubuledisrupting agent, enhance microspore embryogenesis (ME) induction and plant regeneration in bread wheat. To characterize changes in cortical (CMT) and endoplasmic (EMT) microtubules organization and dynamics, associated with ME induction treatments, immunocytochemistry studies complemented by confocal laser scanning microscopy (CLSM) were accomplished. This technique has allowed us to perform advanced 3- and 4D studies of MT architecture. In ME stress induction, the enlargement of the microspore, the nucleus migration to the centre in ‘star-like’ morphology (SLS), and a preprophase band (PPB) formation preceding the symmetric division depends upon the organization and dynamics of both cortical microtubules (CMT) and endoplasmic microtubules (EMT) [16,17,18,19]

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