Elimination of Citrus variegation virus (CVV) by seed cryotherapy

Citrus variegation ilarvirus (CVV) is one of the oldest viruses reported in Citrus, which produces crinkling, puckering and variegation of leaves in field trees and, occasionally, variegated and deformed fruits. This virus is primarily transmitted by grafting. However, no consistent data are available concerning CVV seed transmission, which is apparently low. The elimination of CVV in citrus genotypes is carried out by shoot-tip-grafting. In the case of seed sources, virus elimination directly in the seeds becomes essential. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated by exposing explants to liquid nitrogen. It allows treatment of a high number of samples and results in a high frequency of pathogen-free regenerants. In this work, three polyembryonic Citrus genotypes (sour orange, mandarin and lemon), which were found infected by CVV, were used for trials of virus seed transmission and CVV elimination by dehydration/cryopreservation of the seeds. The dehydrated seeds were placed in cryovials which were then plunged into liquid nitrogen at -196°C. ISSR markers were used to discriminate between zygotic and nucellar plants after treatment. TAS-ELISA and RT-PCR were applied for CVV detection in the seed components before cryopreservation and in the produced seedlings after treatment. CVV seed transmission was assessed for the first time in sour orange and lemon genotypes by serological and molecular means. All tested Citrus genotypes showed CVV infection in both components of the seed plunged in liquid nitrogen. However, all plantlets developed from cryopreserved seeds were CVV-free 2 years after transplanting.