Developing microfluidics for rapid protoplasts collection and lysis from plant leaf

Abstract

This study develops polydimethylsiloxane microfluidics to enable real-time collection and lysis of Phalaenopsis protoplasts and to analyze and compare the protoplast collecting efficiency of a concave sieving array with that of a convex-concave sieving array. Each set of microfluidics comprises a main flow channel and a protoplast sieving array with collecting channels. The protoplasts were isolated from Phalaenopsis leaves by an enzymatic breakdown of the cell walls and collected in side channels through microsieves. Finally, 1% w/v sodium dodecyl sulfate solution was injected into lysed protoplasts, and the DNA segment of the lysed protoplasts was released into the solution. The results indicate that the concave sieving array at the U-bend of the main flow channel causes the protoplasts to flow back into the main flow channel from the collecting channels. Thus, the protoplast collecting efficiency of the concave sieving array was lower in this study. Regarding the number and arrangement of protoplast microsieves, the convex-concave sieving array substantially restricts protoplasts from flowing back into the main flow channel, thereby increasing the collecting efficiency. Finally, DNA is released from lysed protoplasts after the injection of a lysis solution. DNA flow can be focused into a single stream to contact the rectangular microstructures in microchannels.

Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform

“…61 By manipulating the geometry of the microfluidic channels and adjusting the flow rate, it is possible to optimize the protoplast yield from plant cells. 62 Recently, a microfluidicsbased single-cell phenotyping assay has been developed for various Symbiodiniaceae, offering a high-throughput capability to monitor photophysiological changes at the single-cell level under heat stress. 63,64 However, protoplast isolation and monitoring their physiological/photosynthetic capacity in microfluidic chambers have not been established so far.…”

THROUGH THE LOOKING GLASS: Real-Time Imaging in Brachypodium Roots and Osmotic Stress Analysis

“…Those pioneering devices have allowed miniaturisation of the individual experiments and related costs while providing automated parallel assays to achieve accurate as well as high-throughput quantitative data [4,5]. Arabidopsis thaliana [6,7,8,9,10,11,12,13], Camellia Japonica [14,15,16,17], Oryza sativa [18], Nicotiana tabacum [19,20], Phalaenopsis Chiada Pioneer [21], and Physcomitrella patens [22] were the plants that have been employed in various microfluidic platforms for in-depth optical analysis of the dicot seed germination, leaf development, cell phenotypes, protoplasts, pollen tube development and dynamics, shoot and the root growth. As summarized in Table 1, these studies have primarily been carried out in dicot plants and studies on monocot plants are still to be explored.…”

Droplet-based microfluidic analysis and screening of single plant cells

“…Microfluidic devices have been applied for the collection and lysis [ 27 ], culture [ 28 ], chemically-induced fusion [ 29 ], electrofusion [ 30 ], regeneration [ 31 ], and developmental characterization [ 32 ] of plant protoplasts. However, platforms for the high-throughput characterization or sorting of individual plant protoplast based on their level of gene expression have been limited to date.…”