Quantitative real-time PCR analysis of individual flue-cured tobacco seeds and seedlings reveals seed transmission of .
Ellis MD , Hoak JM , Ellis BW , Brown JA , Sit TL , Wilkinson CA , Reed TD , Welbaum G
(TMV) is an extensively studied RNA virus known to infect tobacco ( L.) and other Solanaceous crops. TMV has been classified as a seedborne virus in tobacco. The mechanism of TMV transmission through seed was studied in seed of K 326 cultivar of flue-cured tobacco. Cross pollinations were performed to determine effect of parental tissue on TMV infection in seed. Dissection of individual tobacco seed into seed coat, endosperm, and embryo was performed to determine TMV location within a seed, while 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 non-infected crosses. Results showed maternal transmission of TMV to tobacco seed and seedlings 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 coat of individual seedlings that developed from infected tobacco seed. 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 seeds, demonstrating that TMV is seedborne and seed-transmitted in flue-cured tobacco.
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