Quantitative Real-Time PCR Analysis of Individual Flue-Cured Tobacco Seeds and Seedlings Reveals Seed Transmission of Tobacco Mosaic Virus

Abstract

Tobacco mosaic virus (TMV) is an extensively studied RNA virus known to infect tobacco (Nicotiana tabacum) and other solanaceous crops. TMV has been classified as a seedborne virus in tobacco, with infection of developing seedlings thought to occur from contact with the TMV-infected seed coat. The mechanism of TMV transmission through seed was studied in seed of the K 326 cultivar of flue-cured tobacco. Cross pollinations were performed to determine the effect of parental tissue on TMV infection in seed. Dissection of individual tobacco seeds into seed coat, endosperm, and embryo was performed to determine TMV location within a seed, while germination tests and separation of the developing seedling into seed coat, roots, and cotyledons were conducted to estimate the percent transmission of TMV. A reverse-transcriptase quantitative PCR (RT-qPCR) assay was developed and used to determine TMV concentrations in individual seed harvested from pods that formed on plants from TMV-infected and noninfected crosses. The results showed maternal transmission of TMV to tobacco seed and seedlings that developed from infected seed, not paternal transmission. RT-qPCR and endpoint PCR assays were also conducted on the separated seed coat, endosperm, and embryo of individual seed and separated cotyledons, roots, and seed coats of individual seedlings that developed from infected tobacco seed to identify the location of the virus in the seed and the subsequent path the virus takes to infect the developing seedling. RT-qPCR and endpoint PCR assay results showed evidence of TMV infection in the endosperm and embryo, as well as in the developing seedling roots and cotyledons within 10 days of initiating seed germination. To our knowledge, this is the first report of TMV being detected in embryos of tobacco seed, demonstrating that TMV is seedborne and seed-transmitted in flue-cured tobacco.

Keywords: RT-qPCR; Tobacco mosaic virus; seed transmission; seedborne transmission; virology.