Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic.
IT4Innovations, VSB-Technical University of Ostrava, Ostrava, Czech Republic.
Elife. 2020 Feb 11;9:e52546. doi: 10.7554/eLife.52546.
In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.
在高等植物中，生殖细胞的分化发生在发育中的花朵内相对较短的一段时间内。对控制生殖细胞分化的机制的理解落后于其他植物发育过程。这主要是因为生殖细胞局限于花组织内相对较少的深埋细胞，这使得它们难以研究。为了克服这一限制，我们开发了一种使用光片荧光显微镜对拟南芥花器官中的生殖细胞谱系进行活体成像的方法。我们已经建立了报告基因系、培养条件和成像方案，用于对发育中的花朵进行高分辨率显微镜连续观察长达数天。我们使用多视图成像技术以亚细胞分辨率重建花的三维模型。我们通过捕获雄性和雌性减数分裂、不对称花粉分裂、减数分裂染色体的运动以及绒毡层细胞中的异常有丝后减数分裂，证明了这种方法的有效性。这种方法将为植物有性生殖的研究开辟新途径。
