Abstract:Tobacco flower buds at mid-binucleate pollen stage were cold-treated and their anthers were then float-cultured to release the pollen, which were subsequently macerated in enzyme solution for a short time. And de-exined pollen grains were eventually isolated. The main factors affecting artificial germination of the de-exined pollen were investigated, including osmotic pressure of the enzyme solution during the isolation process, polyethylene glycol (PEG) or sucrose in the culture medium as well as supplementation of lactoalbumin hydrolysate ( LH). Finally, a medium containing D2 macroelements, 30% PEG-6000 and 0.1% LH was established, which supported the de-exined pollen to germinate well with a frequency up to 57.8 %. Mter 24 h of culture, the generative cell in more than half of the pollen tubes, already divided into two sperms. Using a method of micro-sus- pension droplets with the aid of a small piece of filter paper, 30 to 40 de-exined pollen grains were pollinated onto the stigma, resulting in nearly half of the pollen tubes growing in the style and approximately a yield of one seed out of four de-exined pollen grains after subsequent ovary culture. The seeds were germinated into seedlings. The artificial germination of de-exined pollen can be further used as a tool for understanding the role of exine in pollen germination. The in vitro pollination with such exine-free pollen might become a new means for introducing foreign genes into the seeds and offsprings.