Detection of plant virus in meristem by immunohistochemistry and in situ hybridization.

Most plant viruses do not infect the shoot apical meristem (SAM) of a host plant, and this virus-free region of meristem tissue has been used to obtain virus-free clones by meristem tip culture. Thus, the validation of viral distribution in meristem tissues is important for ensuring the appropriate excision of virus-free meristem tips. Although immunohistochemical microscopy and in situ hybridization are classical techniques, they allow us to determine the presence or absence of plant viruses in the shoot meristem tissues of a host plant. Briefly, meristem tissues are excised from infected plants, fixed, embedded in paraffin medium, and prepared in semithin sections (10-15 μm). By treating these sections with an antibody against viral protein or with a probe complementary to viral RNA, the viral distribution in the meristem tissue can be clearly observed. Importantly, these procedures are broadly applicable to most virus (and viroid) and host plant combinations.