Abstract
Microspore cultures generate a heterogeneous population of embryogenic structures that can be grouped into highly embryogenic structures [exine-enclosed (EE) and loose bicellular structures (LBS)] and barely embryogenic structures [compact callus (CC) and loose callus (LC) structures]. Little is known about the factors behind these different responses. In this study we performed a comparative analysis of the composition and architecture of the cell walls of each structure by confocal and quantitative electron microscopy. Each structure presented specific cell wall characteristics that defined their developmental fate. EE and LBS structures, which are responsible for most of the viable embryos, showed a specific profile with thin walls rich in arabinogalactan proteins (AGPs), highly and low methyl-esterified pectin and callose, and a callose-rich subintinal layer not necessarily thick, but with a remarkably high callose concentration. The different profiles of EE and LBS walls support the development as suspensorless and suspensor-bearing embryos, respectively. Conversely, less viable embryogenic structures (LC) presented the thickest walls and the lowest values for almost all of the studied cell wall components. These cell wall properties would be the less favorable for cell proliferation and embryo progression. High levels of highly methyl-esterified pectin are necessary for wall flexibility and growth of highly embryogenic structures. AGPs seem to play a role in cell wall stiffness, possibly due to their putative role as calcium capacitors, explaining the positive relationship between embryogenic potential and calcium levels.
Highlights
IntroductionDuring the development of the male gametophyte (microsporogenesis and microgametogenesis), cells go through several stages before becoming mature, functional pollen grains
During the development of the male gametophyte, cells go through several stages before becoming mature, functional pollen grains
As it was not clear whether the presence of compact callus (CC) and loose callus (LC) in microspore cultures is associated with the recalcitrance of this genotype, we reexamined the types of structures found in DH4079 cultures in quantitative (Figure 1A) and qualitative detail (Figures 1B–E)
Summary
During the development of the male gametophyte (microsporogenesis and microgametogenesis), cells go through several stages before becoming mature, functional pollen grains. Microspores grow and mature and eventually divide asymmetrically to give rise to pollen grains, within which the male gametes will be produced This natural pathway can be altered when microspores/pollen are cultured in vitro. In B. napus and many other species, microspore embryogenesis is triggered by the application of a heat stress treatment to isolated microspores This treatment is associated with a series of cellular changes at different levels, involving the cytoskeleton, nucleus, cytoplasm, organelles, and cell wall (Simmonds and Keller, 1999; Seguí-Simarro and Nuez, 2008; Seguí-Simarro et al, 2011; Corral-Martínez et al, 2013; Parra-Vega et al, 2015a; Rivas-Sendra et al, 2019)
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