Allan Claudia, Elliot Blake, Nock Volker, Meisrimler Claudia-Nicole
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
Bio Protoc. 2023 Aug 5;13(15):e4764. doi: 10.21769/BioProtoc.4764.
Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress-signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual-flow-RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as , , and , on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved. Key features Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments. Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 μm width, and 500 μm height. Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines. Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC. Graphical overview (a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.
由于技术限制，迄今为止的研究主要集中在非生物和生物胁迫信号分子在植物地上器官中的作用，包括整个地上部分、茎和叶。此后，包括双流根芯片（dfRC）、植物芯片和根阵列在内的新型实验平台已将此扩展到植物根细胞分析。基于用于对顶端生长生物体进行流场塑造和力传感的微流控平台，dfRC进一步扩展为双向双流根芯片（bi-dfRC），并入了第二对相邻的入口/出口，能够对植物根进行双向不对称灌注处理（从地上到根或从根到地上）。本方案详细概述了bi-dfRC平台的设计和使用。芯片上的植物培养与引导根生长以及使初生根受控暴露于溶质变化相结合。可以追踪表面处理对根生长和防御信号的影响，以应对非生物和生物胁迫或两者的组合效应。特别是，本方案突出了该平台在芯片上培养多种植物（如 、 和 ）的能力。它表明，通过简单改变bi-dfRC的尺寸，在微流控条件下实现了研究具有不同初生根大小的所需植物物种的广泛应用基础。关键特性 将斯坦利等人（2018a）开发的用于研究对局部处理作出反应的防御信号方向性的方法进行扩展。描述了一种微流控平台，该平台允许培养初生根长度达40毫米、宽度达550微米、高度达500微米的植物。用聚乙烯吡咯烷酮（PVP）处理以永久保持部分疏水的bi-dfRC微通道的亲水性，从而能够用于对表面敏感的植物品系。描述了配备可旋转阀的新型管道阵列设置，用于在bi-dfRC上进行实时成像时切换处理试剂和方向。图形概述 （a）描绘光刻和复制模塑以生产聚二甲基硅氧烷（PDMS）装置的示意图。（b）描绘芯片外种子培养，然后将4日龄幼苗在芯片上继代培养的示意图。（c）描绘显微镜和成像设置的示意图，配备用于在不同条件下将不对称处理引入bi-dfRC微通道进行根生理分析的培养基输送系统。
