Establishment and maintenance of Soybean Mosaic Virus in soybean callus culture

Abstract

Susceptibility to inoculation with Soybean mosaic virus (SMV) and virus activity were investigated in soybean callus cultures growing in vitro. Excised hypocotyls of susceptible soybean, Glycine max (L.) Merr. ‘Essex’, were cultured in Msoy medium in the light at 25 °C. Established calluses were inoculated with SMV in vitro by a soak–prick method. In addition, SMV-infected leaves of soybean ‘Lee 68’ were surface sterilized, excised, and placed on callus-inducing medium. Calluses infected with SMV initiated by either method grew in vitro as well as calluses from uninfected tissues. Callus cultures turned brownish yellow after 6–8 weeks, when the media became depleted of nutrients. However, callus with an active SMV infection could be maintained by regular subculture to fresh medium. Longevity of SMV–callus cultures was increased by storage at 10–15 °C, thus reducing the frequency of transfers. The virus was detected in infected calluses by serological tests. Infectivity assays confirmed the presence and viability of SMV in callus cultures. Most callus cultures induced directly from infected leaves retained virus and high infectivity, whereas infective cultures from in vitro inoculated hypocotyls appeared to decrease in number and infectivity with repeated subcultures. However, selected infective cultures retained high infectivity after 10 successive transfers over a period of 20 months. These results demonstrate that SMV can be cultured and maintained active in callus cultures in vitro