Massively Parallelized Pollen Tube Guidance and Mechanical Measurements on a Lab-on-a-Chip Platform

Abstract

Pollen tubes are used as a model in the study of plant morphogenesis, cellular differentiation, cell wall biochemistry, biomechanics, and intra- and intercellular signaling. For a “systems-understanding” of the bio-chemo-mechanics of tip-polarized growth in pollen tubes, the need for a versatile, experimental assay platform for quantitative data collection and analysis is critical. We introduce a Lab-on-a-Chip (LoC) concept for high-throughput pollen germination and pollen tube guidance for parallelized optical and mechanical measurements. The LoC localizes a large number of growing pollen tubes on a single plane of focus with unidirectional tip-growth, enabling high-resolution quantitative microscopy. This species-independent LoC platform can be integrated with micro-/nano-indentation systems, such as the cellular force microscope (CFM) or the atomic force microscope (AFM), allowing for rapid measurements of cell wall stiffness of growing tubes. As a demonstrative example, we show the growth and directional guidance of hundreds of lily (Lilium longiflorum) and Arabidopsis (Arabidopsis thaliana) pollen tubes on a single LoC microscopy slide. Combining the LoC with the CFM, we characterized the cell wall stiffness of lily pollen tubes. Using the stiffness statistics and finite-element-method (FEM)-based approaches, we computed an effective range of the linear elastic moduli of the cell wall spanning the variability space of physiological parameters including internal turgor, cell wall thickness, and tube diameter. We propose the LoC device as a versatile and high-throughput phenomics platform for plant reproductive and development biology using the pollen tube as a model.

3D Manipulation and Imaging of Plant Cells using Acoustically Activated Microbubbles

“…Next, we demonstrate the rotation of Arabidopsis thaliana pollen grains, which are much smaller and more spherical than those of lily. Although these grains are ≈4 to 5 times smaller than lily pollen grains, the same device geometry can be utilized for their 3D rotational manipulation. Figure a shows a single pollen grain rotating in‐plane (Movie S5, Supporting Information).…”

“…The growth medium for lily pollen was prepared with the following components: 160 m m H 3 BO 3 , 130 m m Ca(NO 3 ) 2 , 1 m m KNO 3 , 5 m m MES, and 10% sucrose. The rehydrated lily pollen grains were measured to have a major and minor axis of 112.5 ± 8.9 µm and 92.4 ± 8.5 µm, respectively ( n = 40) …”

“…The growth medium for the Arabidopsis pollen was prepared with the following components: 1.6 m m H 3 BO 3 , 5 m m CaCl 2 , 5 m m KCl, 1 m m MgSO 4 , 10% sucrose, and 1% low melting agarose at pH 7.5. The major and minor axis of the Arabidopsis pollen grains were found to be 26.7 ± 2.4 µm and 20.0 ± 1.3 µm, respectively ( n = 40) …”

“…Plant cells, such as the pollen grains and pollen tubes, play a key role in plant sexual reproduction, and serve as model systems for cellular morphogenesis. Pollen, also known as the male gametophyte of flowering plants, carries and transports the two sperm cells within its cytoplasm.…”

Nano‐engineering pollination

“…Using a micro‐indentation system, Shamsudhin et al . () assessed the apparent cell wall stiffness of pollen tubes, c . 2 N m −1 .…”