A method for loading fluorescent dyes in pollen tubes

Abstract

The invention discloses a method for loading fluorescent dye into a pollen tube. The method comprises a first step of coating pollen with weighing paper and placing the pollen in a closed silica gel bottle to be stored in a low temperature refrigerator; a second step of taking out the pollen from the refrigerator, standing the pollen for 2 hours at normal temperature, adding the pollen into nutrient solution, evenly stirring the solution and then placing the solution into a thermostat of 25 DEG C to perform cultivation for 2 hours; and a third step of correspondingly mixing the nutrient solution, lysis solution and a coloring agent, being in the absence of light for 10-30 minutes and placing the mixture in the thermostat of 25 DEG C for cultivation for 2 h in the absence of light. According to the method, the lysis solution is adopted to process and damage cytomembrane of the pollen, so that the coloring agent can quickly and effectively enter cells. The lysis solution and the coloring agent (Fluo-4AM-ester) are utilized to process the pollen tube for 20 minutes at normal temperature (25 DEG C), and calcium ions in the pollen tube can be dyed successfully.

Description

technical field [0001] The invention relates to the field of polar growth of pollen tubes, in particular to a method for loading fluorescent dyes in pollen tubes. Background technique [0002] The normal growth of pollen tubes is the result of the regulation of various signaling molecules, in which calcium ions play an important role. Higher calcium gradients at the cytoplasmic tips of pollen tubes have been observed in a variety of plants, and the disappearance of the calcium concentration gradient leads to arrest of pollen tube growth. The high calcium gradient at the top of the pollen tube may have the following functions: first, it may be involved in the regulation of vesicle secretion, because a high concentration of calcium facilitates the fusion of the secretory vesicle and the plasma membrane, promotes efflux, and extends the pollen tube; second , high concentration of calcium depolymerizes microfilaments and slows down the movement of organelles, so the calcium gra...

AI Technical Summary

Problems solved by technology

The loading methods of calcium ion fluorescent indicators include: electroporation (this method causes great damage to the cells, and the equipment is expensive, and is generally not used now), microinjection (this method has a certain degree of damage to the cells, and the operation is complicated and the workload is high. Large size, low success rate, not suitable for small individual cells, not suitable for batch cells and other shortcomings limit the wide application of this method, patch clamp (slow speed, expensive equipment), low temperature method (long time), High temperature method (affects survival)