Distribution of Tomato planta macho viroid in germinating pollen and transmitting tract

Vertical and horizontal pollen transmission is important for efficient infection by viroids. Vertical pollen transmission of viroids is attributed to the infection by viroid in the embryo sac through infected pollen. To identify the viroid infection in pollen and pollen tubes elongating through the transmitting tract, we used in situ hybridization to histochemically analyze the distribution of Tomato planta macho viroid (TPMVd) in pollen grains, the stigma, and style of petunia plants. TPMVd was present in the generative nucleus and vegetative nucleus of mature infected pollen grains and germinating pollen grains. During pollen tube growth, TPMVd was present in the vegetative nucleus and two sperm nuclei, which were generated by division of the generative nucleus in the style transmitting tract. These findings indicated that viroid infection in sperm nuclei is responsible for vertical pollen transmission of viroids. TPMVd infection from TPMVd-infected pollen tubes to the transmitting tract was not observed. In addition, TPMVd signals were not confirmed in the stigma and transmitting tract of TPMVd-infected petunia plants, suggesting that viroids may not replicate in these tissues at the stage of mature style. Therefore, TPMVd may leak from the pollen tube somewhere in the ovary, except in the transmitting tract, during the horizontal transmission of TPMVd.