Abstract:
Abstract: The plant immune elicitors can induce a line of defense responses in plants and are identifed as new type of biological pesticides. At present stage, new plant immune elicitor is screening with potted plants or field experiments, which is time-consuming and high-cost. The droplet microfluidic technology, which is originated from analytical chemistry and owns micro-channel network structure, has properties of high throughput, high sensitivity, low consumption of reagent, no cross contamination and rapid reaction. These advantages provide a novel platform for screening plant immune elicitors. Chitosan oligosaccharides (COS) are obtained by degradation of chitosan. It was reported that COS could activate plant innate immunity, such as: stimulate H2O2 (hydrogen peroxide) production, induce defense response by NO (nitric oxide) pathway, make a synthesis of phytoalexin, impact the jasmonic acid / ethylene (JA/ET) signaling marker, trigger defense-related gene expression, cause changes in protein phosphorylation, activate mitogen-activated protein kinases (MAPKs), and possess antimicrobial activity against bacteria and fungi in plant. Because of the advantages and the high solubility, nontoxicity, and biocompatibility, COS are considered as an effective plant immune elicitor by researchers. To preliminarily applying droplet microfluidic technology in plant immune elicitor screening, integrated microfluidic chip with droplets formation structure was designed and fabricated. COS were chosen as positive reagent, and BY-2 tobacco cells played as model plant cell. The flow rate of mobile phase was measured and established for BY-2 tobacco cells droplets formation, and the single cell encapsulating efficiency was calculated. Then COS and NO probe were dumped into the droplets with BY-2 tobacco cells, and the fluorescence intensity of NO probe from droplets was detected to evaluate the feasibility of screening the plant immune elicitor COS. To make the comparative analysis, the fluorescence intensity was compared with the same reaction system in 96-well plate. The results showed that at the flow rates of 100 μL/h in cell suspension and 300 μL/h in oil, the sizes of generated droplets were suitable to encapsulate the cells. The BY-2 cell clusters could be dispersed in isotonic solution, and every droplet encapsulated single cell. The ratio of droplets encapsulating single cell was about 22.9%. The ratio conformed to Poisson distribution. The fluorescence intensity of droplets incubated with COS was detected by fluorescence microscope. The fluorescence intensity from the COS/NO probe/BY-2 cell group was significantly higher than the control groups. In the comparative analysis experiments, the fluorescence intensity of droplets showed similar trend compared with the same reaction system in 96-well plate. This result implied that the cell treated with COS in droplets showed similar trend in defense responses compared to traditional screening method with 96-well plate. Thus, the droplets with COS / NO probe / BY-2 cell show high fluorescence intensity that can be detected by fluorescence microscope, which implies that integrated with fluorescence detecting technique, droplets microfluidics and fluorescence probe can be a novel platform for screening plant immune elicitors.