Observational experiments on pollen germination and pollen tube growth
Source: Experiments in Botany
Operation method
basic program
Principle
Pollen is the male gametophyte of seed plants, and it is produced in the anthers of the stamens. During sexual mating, pollen grains carrying the genetic information of the parent are deposited on the stigma of the female via insect or wind pollination, and the pollen sprouts pollen tubes that carry the sperm produced in the male gametophyte to the female gametophyte to complete fertilization. Although angiosperms and gymnosperms pollen grains and their placement of the female structure are very similar, but the pollen germination and pollen tube children long is the process of sexual reproduction must undergo. Viable pollen and pollen germination on female structures, pollen tube growth and competition between pollen tubes are the prerequisites for the production of a sufficient number of potentially adaptable offspring. In vitro germination is used to understand pollen viability by detecting the pollen germination rate, and fluorescent dyes can be used to stain pollen tubes, so that the pathways for the growth of pollen tubes in the female reproductive organs can be observed under a fluorescent microscope.
Materials and Instruments
pollen Move 1 In vitro test of pollen germination In the in vitro test of pollen germination and pollen tube elongation, the commonly used culture is sucrose solution or agar medium containing sucrose. Although the concentration of sucrose required for pollen germination varies from plant to plant, it is generally 2%-20%, and the pollen of some aquatic plants has a higher germination rate in distilled water. Adding 0.01% boric acid to the culture medium can promote the germination of pollen of most plants. Some plants have better pollen germination only on gelatin medium with sucrose. When liquid medium is used, pollen germination is not counted due to pollen settling, so the droplet culture method can be used, or cellophane can be used as a support for pollen germination. When agar medium is used, the culture some is poured on a concave slide or in a small petri dish to enable observation under a microscope. In the study of pollen germination, the medium should first be prepared using a combination of different concentrations of sucrose and boric acid, from which the appropriate formulation should be selected. After the pollen grains are placed in the medium, they should be selected to be incubated at the optimum temperature' Generally, pollen can be observed to sprout from pollen tubes after tens of minutes to a few hours of incubation on the medium, and the experimental procedure should be determined to start counting the germination rate after a consistent period of incubation. -Generally, pollen tubes with a length more than 2 times of the pollen diameter are considered to have sprouted, and the percentage of sprouted pollen in the total number of pollen in the field of view is calculated, 20 or more fields of view are suitable for each group of experimental materials. 2 Fluorescence microscopic observation of pollen tube growth pathway Fluorescence microscopy is often used in the study of pollen grains and pollen tubes. The fluorescent dye aniline blue can stain the pollen tube walls and callus plugs of pollen tubes, while bright yellow or yellow-green under ultraviolet or basket violet light. The usual procedure for observing the pollen tube callus in the pistil of angiosperms is as follows: the pollinated pistil is fixed with FAA fixative, 5% potassium hydroxide or sodium hydroxide is dissolved and transparent to a moderate degree (usually takes 1h or more), rinsed three times with distilled water, take an appropriate amount of material on a slide, add 1-2 drops of aniline blue stain (0.lg of water-soluble aniline blue, dissolved in 1/30 mol/L potassium phosphate) to 100mL, keep away from light, and then add 1-2 drops of aniline blue stain (water-soluble aniline blue 0.lg, dissolved in 1/30 mol/L potassium phosphate). (100mL, keep under refrigeration and protect from light until nearly colorless.) Stain for about 1h, add 1 drop of 50% glycerol, cover the coverslip, and apply light pressure to the camera. The slide made after this treatment was observed with a fluorescence microscope, and the pollen tubes in the pistil tissue showed strong fluorescence under the basket violet light, which was very easy to observe. In addition, without the use of fluorescence microscope, there are also only pollen tube staining method: after pollination of the pistil in alcohol - trichloromethyl acetate - acetic acid (3:4:1) solution fixed for 30min, in 70% alcohol, 50% alcohol, 30% alcohol, distilled water, one by one in alcohol immersion for 2min and then removed, with aniline blue - sapphire red O staining solution (water). The flowers were stained with aniline blue and saffron red O staining solution (0.75g of water-soluble aniline blue and 00.25g of saffron red, dissolved in heated 45% acetic acid and filtered), and then transparent in 45% acetic acid solution for 1 h, and then blocked with lactic acid phenol. Under the microscope, the stylar tissue stained very light purple, while the pollen tube stained dark blue, which was easy to distinguish. For more product details, please visit Aladdin Scientific website.
Dye
Petri dishes