FWO Sabbatical professor Danny Geelen
Microspore regeneration is induced by subjecting immature flowers to specific stress treatments, such as cold, heat, or starvation, after which microspores are isolated and cultured in vitro. Given the haploid nature of the microspore, a chromosome-doubling step is necessary to regenerate into a fertile, DH plant. Every flower produces thousands of microspores with the potential to be switched into androgenesis. However, at every step of the procedure, many microspores are lost, ultimately yielding very few DH lines for breeding. A minimum of 1 embryo regenerated from 1000 microspores is a rule of thumb limit for industrial application. This has only been achieved for a handful of crops (barley, rapeseed, broccoli, cauliflower and tobacco).
The competence of microspores to regenerate and to initiate embryogenesis can be exploited to save multiple generations per breeding cycle. There are, however, two key problems with regeneration. Firstly, only a fraction of the cells is competent, and many do not regenerate or even inhibit regeneration of others. This problem can be solved by separating cells with high competence from non-competent ones. Secondly, the conditions under which cells engaging in regeneration divide and form cell clumps (micro calli) that produce embryos, are suboptimal, and are largely determined empirically. A general approach to address these issues is presented in an SBO2020 research project proposal (PASCell) that was submitted in October 2020.
Microspores are susceptible to a stress stimulus from the mid-to-late uninuclear stage. The androgenesis-competent cells are highly vacuolated with an eccentric nucleus. Microspores at a later stage in development are usually less competent, albeit that variability is observed in different species. During meiosis, the pollen mother cell (PMC) is surrounded by a temporary wall consisting of mainly callose that is degraded before the pollen wall develops. The formation of the pollen wall is a stepwise process initiated with the formation of the primexine, a cellulosic cell wall that provides the support for the deposition of sporopollenin, proteinaceous cell wall material that is generated by the surrounding tapetum cell layer. However, in the context of microspore regeneration, the cell wall develops differently in response to the temperature stress applied and the in vitro culturing. It involves the deposition of a cellulosic layer that is not formed during regular development of the microspore.
Since cell wall properties are likely having an impact on the U+201CstiffnessU+201D of microspores, I tested in preliminary experiments whether microspores can be analyzed using atomic force microscopy. In collaboration with Prof. Andre Skirtach (UGent, faculty of Bioscience Engineering), I found that microspores from separate barley inflorescences samples display differential stiffness (not shown). Moreover, microspores from flowers that were heat stressed required 5 times more force to deform. These results suggest that mechanical properties of microspores are putatively correlated with the stress and possibly development stage. Further studies will reveal more details on the relationship with cell wall development and responses to stress.