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.